Stat3 controls lysosomal-mediated cell death in vivo

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1 L E T T E R S Stt3 controls lysosoml-medited cell deth in vivo Peter A. Kreuzler 1, Ann D. Stniszewsk 1, Wenjing Li 1, Nder Omidvr 2, Blndine Kedjour 1, Jmes Turkson 3, Vleri Poli 4, Richrd A. Flvell 5, Richrd W. E. Clrkson 2 nd Christine J. Wtson 1,6 It is well estlished tht lysosomes ply n ctive role during the execution of cell deth 1. A rnge of stimuli cn led to lysosoml memrne permeiliztion (LMP), thus inducing progrmmed cell deth without involvement of the clssicl poptotic progrmme 2,3. However, these lysosoml pthwys of cell deth hve mostly een descried in vitro or under pthologicl conditions 4 7. Here we show tht the physiologicl process of post-lcttionl regression of the mmmry glnd is ccomplished through non-clssicl, lysosoml-medited pthwy of cell deth. We found tht, during involution, lysosomes in the mmmry epithelium undergo widespred LMP. Furthermore, lthough cell deth through LMP is independent of executioner cspses 3, 6 nd 7, it requires Stt3, which upregultes the expression of lysosoml proteses cthepsin B nd L, while downregulting their endogenous inhiitor Spi2A (ref. 8). Our findings report previously unknown, Stt3-regulted lysosoml-medited pthwy of cell deth under physiologicl circumstnces. We nticipte tht these findings will e of mjor importnce in the design of tretments for cncers such s rest, colon nd liver, where cthepsins nd Stt3 re commonly overexpressed nd/or hyperctivted respectively 1,9,1. Apoptosis is often used synonymously with the term progrmmed cell deth, ecuse mny of the min cell deth events hve een identified s such. However, more recent work hs shown tht, prticulrly under circumstnces in which cells hve ecome refrctory to poptosis, other pthwys leding to progrmmed cellulr demise cn e found 4. In this context, the lysosomes ply crucil role nd it is now well estlished tht prtil lysosoml memrne permeiliztion (LMP) leds to relocliztion of lysosoml components, such s cthepsins, to the cytosol, where they cn ct s executioner proteses 11. Furthermore, n incresing numer of stimuli including rective oxygen species, clpins, sphingosine nd the deth receptor lignds tumour-necrosis fctor (TNF)α, Fs nd TNF-relted wek inducer of poptosis (TWEAK) hve een identified s LMP-inducing gents 11. Nevertheless, the lysosoml-medited pthwy of cell deth is still frequently elieved to e just ckup mechnism when clssicl poptotic stimuli fil to induce cell deth. Post-lcttionl regression (involution) of the mmmry glnd epithelium is one of the min cell deth events in the dult mmmlin orgnism. In rodents, the complete involution of the mmmry glnd to ner pre-pregnnt stte is ccomplished within 1 dys fter wening 16. Studies over the pst decde using geneticlly engineered mice hve reveled criticl upstrem regultors of involution. These include solule signlling molecules such s trnsforming growth fctor-β 3 (TGFβ3) nd leukemi inhiitory fctor (LIF), trnscription fctors such s Stt3 nd CCAAT/enhncer inding protein-δ (Cepδ) nd remodelling enzymes such s mtrix metlloproteses However, lthough presumed to e poptosis, the modlity of progrmmed cell deth (PCD) during involution is not known. To investigte the mechnism of PCD in vivo, we crried out morphologicl nlysis of mmmry glnd involution in mice. This reveled severl fetures tht were typicl for clssicl poptosis, such s two hypercondensed nuclei, swelling of the cells nd complete lck of memrne leing, while showing no fetures of recently descried form of PCD termed entosis, tht hs een discovered in mmmry epithelil cells in vitro 23 (Fig. 1). Furthermore, TdT-medited dutp nick end lelled (TUNEL) positive cells could not e detected in the first, reversile, phse of involution ( 2 dys fter wening), wheres in the second, remodelling phse strong TUNEL stining ws oserved (Fig. 1 nd Supplementry Fig. S1). Despite trits of non-clssicl poptotic PCD, executioner cspses re ctivted nd cytochrome c is relesed from mitochondri during involution 24. Closer inspection, however, reveled temporl hierrchy of cspse ctivtion in which cspses 3 nd 6 re ctivted in oth the first nd second phses, wheres ctive cspse 7 ws exclusively detected in the irreversile phse (Fig. 1c). Furthermore, in erly involution, cleved cspses 3 nd 6 could e detected only in cells tht hd lredy een shed from the lveolr epithelium, wheres not single cell positive for cleved cspse 7 could e detected efore dy 3 of involution (Fig. 1d). To determine whether these cspses re required for PCD during this phse, we generted cspse 3 nd 6 douly 1 Deprtment of Pthology, University of Cmridge, Tennis Court Rod, Cmridge CB2 1QP, UK. 2 School of Biosciences, University of Crdiff, Crdiff CF1 3US, UK. 3 University of Centrl Florid, Reserch Prkwy, Orlndo, Florid 32826, USA. 4 The Moleculr Biotechnology Center (MBC), Deprtment of Genetics, Biology nd Biochemistry, University of Turin, 1126 Turin, Itly. 5 Section of Immunoiology, Yle University School of Medicine, New Hven, Connecticut 65, USA. 6 Correspondence should e ddressed to C.J.W. (e-mil: cjw53@mole.io.cm.c.uk) Received 8 Novemer 1; ccepted Decemer 1; pulished online Ferury 11; DOI: 1.138/nc2171 NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

2 L E T T E R S d Erly PCD TUNEL/Hoechst Lte PCD 1 d involution 3 d involution c M r (K) cc 3/Hoechst cc 6/Hoechst cc 7/Hoechst * Wild type C3 KO d lc d involution Wild type C6 KO d lc d involution Wild type C7 KO d lc d involution cc 7/Hoechst pro-c 3 cc 3 p19 cc 3 p17 β-ctin pro-c 6 cc 6 β-ctin pro-c 7 cc 7 β-ctin 1 dy involution 3 dys involution Figure 1 Chrcteristics of physiologicl cell deth in mmmry glnd involution. () Representtive trnsmission electron microgrphs of shed mmmry epithelil cells in tissue from 1 dy involution time point t erly nd lte stges of PCD, showing no leing (rrows) nd hypercompcted nuclei (rrowheds; scle rs, 2 µm; the sterisk mrks ft droplet). () TUNEL stining of mmmry glnd sections t 1 dy nd 3 dys involution. At 1 dy involution ll cells including shed cells (rrowheds) re TUNEL negtive. At 3 dys involution mny shed cells (rrowheds) nd cells in the lveolr wll (rrows) re TUNEL positive (scle rs, µm). (c) Representtive western lot nlysis of t lest three independent iologicl smples showing pro- nd cleved cspses 3, 6 nd 7 (pro-c nd cc 3, 6 nd 7, respectively) during lcttion (lc) nd involution with respective cspse-knockout (KO) tissue controls. Differentil ctivtion of the executioner cspses is pprent. (d) Immunohistochemicl nlysis of the occurrence of cleved executioner cspses 3, 6 nd 7 (cc 3, 6 nd 7, respectively) during mmmry glnd involution. Cleved cspses 3 nd 6 cn e detected in erly involution, ut re restricted to shed cells, wheres cleved cspse 7 is detected only strting from 3 dys of involution. Immunostining of single lveolus is shown for ech imge. Scle rs, µm. Uncropped imges of lots re shown in Supplementry Fig. S9. deficient mice (C3+6-knockout). Compenstory ctivtion of cspse 7 did not occur in these mice, providing us with system in which no executioner cspse ws ctive (Fig. 2 c). Surprisingly, involution progressed unted, leding us to conclude tht the cell deth process in erly involution is unlikely to rely on the ctivtion of executioner cspses (Fig. 2d nd Supplementry Fig. S2). These findings were recpitulted in doxycycline-inducile doule-trnsgenic mouse strin overexpressing the virl cspse inhiitor p35 in the mmmry glnd under control of the mouse mmmry tumour virus promoter (Fig. 2e nd Supplementry Fig. S2). Given the unusul morphology of PCD during involution s well s executioner cspse independence, we investigted lterntive cell deth mechnisms. We oserved striking downregultion of the structurl lysosoml memrne protein LAMP2 in the mmmry glnd during lcttion tht persisted until the second dy of involution (Fig. 3). At trnscriptionl level, oth Lmp2 nd Lmp1 messenger RNAs re downregulted during lcttion (Supplementry Fig. S3). Diminished LAMP expression hs een suggested to sensitize cells to cell deth y lysosoml lekge, which prompted us to investigte lysosoml-medited PCD (LM-PCD; ref. 26). The cysteine cthepsins B nd L were mrkedly upregulted with the onset of involution, which consequently trnslted into striking increse in their ctivity (14-fold nd 16-fold respectively; Fig. 3, nd Supplementry Fig. S4). This is in contrst to the sprtic cthepsin D, which hs een shown to e only modertely ctive in the first phse of involution 27. Immunohistochemicl nlysis reveled tht, lthough cthepsin B minly co-loclized with LAMP2 during lcttion, with the onset of involution this co-locliztion dropped shrply, which ws reflected in decresed Mnder s coefficient (Fig. 3c,d). To directly mesure cthepsin ctivity in the cytoplsm of mmmry epithelil cells during involution, we crried out sucellulr frctiontion of mmmry tissue from lcttion nd involution time points. Strikingly, during lcttion, over 95% of the totl cthepsin B ctivity ws found in the lysosoml frction, ut fter 34 NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

3 L E T T E R S M r (K) C3+6 knockout Dys involution pro-c 7 d C3+6 knockout cc 7 β-ctin 1 d involution Fluorescence units d inv 2 d inv 3 d inv 1 d inv 2 d inv 3 d inv 3 d involution e p35; Dox p35; Dox+ C3+6 knockout c Fluorescence units d inv 2 d inv 3 d inv 1 d inv 2 d inv 3 d inv 1 d involution 3 d involution C3+6 knockout Figure 2 Cell deth in erly involution is independent of executioner cspses. () Western lot nlysis of cspse 7 in C3+6-knockout mice nd wild-type controls during involution confirms lck of compenstory ctivtion of cspse 7 in erly involution. (,c) Cspse ctivity ws mesured with the fluorescent sustrtes AC-DEVD-AMC for cspses 3 nd 7 () nd AC-VEID-AMC for cspse 6 (c). In the cspse 3 nd 6 doule-knockout mice, no compenstory cspse 7 ctivtion could e mesured. The grphs show representtive time course; ll vlues re mens ± s.d. of three independent mesurements. (d) Unted progression of mmmry glnd involution in C3+6-knockout nd wild-type control mice, shown on hemtoxylinnd eosin-stined sections (scle r, µm). d were crried out for five independent iologicl smples. (e) Unted progression of mmmry glnd involution in p35-overexpressing mice fter induction of the trnsgene y doxycycline, shown on hemtoxylinnd eosin-stined sections representtive of t lest three independent iologicl repets (scle r, µm). Uncropped imges of lots re shown in Supplementry Fig. S9. only 24 h involution the ctivity ws eqully distriuted etween the cytoplsmic nd lysosoml frctions. Cthepsin L ctivity ws considerly incresed, in ddition to eing redistriuted to the cytoplsm during involution, with the lower cytosolic-to-lysosoml rtio, compred with cthepsin B, most proly eing due to higher de novo synthesis of cthepsin L t this time point (Fig. 3e nd Supplementry Fig. S4,c). It is well estlished tht cthepsins cn not only lek into the cytosol, ut lso cn e ctively relesed into the intercellulr spce to degrde the extrcellulr mtrix 28. To ensure tht the cthepsin ctivity in the cytosolic frction did not merely originte from the extrcellulr spce, we mesured the lekiness of purified mmmry lysosomes y incution in neutrl uffer in vitro. During lcttion, lysosomes lost very little cthepsin B nd L, ut with the onset of involution there ws n lmost complete loss of cthepsins from lysosomes within 9 min (Fig. 3f). This correlted with higher cthepsin B nd L ctivity in the incution uffer (Supplementry Fig. S5). Thus, during mmmry glnd involution, there is mrked increse in LMP, resulting in exodus of cthepsins B nd L into the cytoplsm. We hve shown previously tht Stt3 is essentil for mmmry epithelil cell PCD nd susequent involution 19 (Fig. 4 nd Supplementry Fig. S6,). Using conditionl Stt3 fl/fl;blg-cre mice, in which Stt3 is deleted exclusively from the mmmry epithelium during lcttion (herefter clled Stt3-knockout), we oserved tht during involution the protein nd mrna levels of oth cthepsins B nd L were considerly lower thn time-mtched Stt3 fl/fl controls (herefter clled Stt3 controls; Fig. 4,c). Moreover, sucellulr frctiontion reveled tht the ctivity of cytosolic cthepsins B nd L ws 12 nd seven times lower in the Stt3-knockout tissue respectively t 1 dy involution nd remined diminished throughout involution in oth frctions (Fig. 4d,e). Conversely, stimultion of EpH4 mmmry epithelil cells with oncosttin M (OSM), cytokine tht ctivtes Stt3 during involution, led to mrked increse in cthepsin B nd L protein levels nd more widespred cellulr locliztion 29 (Fig. 4f,g). When co-incuting these cells with cycloheximide, n ccumultion of cthepsin mrna could e detected, rguing for trnscriptionl control downstrem of OSM without the need for protein synthesis (Fig. 4h). Furthermore, stining these cells with the lysosomotropic dye Lysotrcker Red showed tht fter tretment with OSM, EpH4 cells undergo LMP, s cn e seen y the ppernce of popultion of cells with low Lysotrcker intensities (Fig. 4i,j). Tken together, this shows the direct involvement of Stt3 in the regultion of the expression of cysteine cthepsins nd their locliztion. NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

4 L E T T E R S M r (K) 1 Dys lcttion Dys involution LAMP2 pro-cts B sc cts B dc cts B pro-cts L sc cts L β-ctin Fluorescence units d lc Cthepsin B Cthepsin L.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv d M green d lc 1 d inv 2 d inv c cts B/LAMP2/Hoechst 1 1 d lc 1 d inv 2 d inv e Fluorescence units Cthepsin B Lysosoml Cytosolic 1 d lc 1 d inv f M r (K) 1 1 dys lcttion 1 dy involution Pellet Superntnt Pellet Superntnt M M min t C Figure 3 Mmmry lysosomes ecome leky during involution. () Western lot showing decrese of LAMP2 during lcttion nd erly involution nd increse of cthepsin B nd L (cts B nd L; sc, single chin, dc, hevy chin of the doule-chin form; oth the single-chin nd the doule-chin form re ctive) in involution. () Incresing cthepsin B nd L ctivity in whole glnd lystes during lcttion nd involution ws mesured y clevge of the fluorescent sustrte Z-Phe-Arg-AMC. Error rs represent the stndrd devition of mesurements from three independent iologicl smples. (c) Immunohistochemicl nlysis of the sucellulr locliztion of cthepsin B (green) with respect to the lysosoml mrker LAMP2 (red) in lcttion nd involution time points. A decresed co-locliztion during involution is pprent. Nuclei stined with Hoechst (scle rs, µm). (d) Mnder s coefficient of co-locliztion for cthepsin B (green) with respect to LAMP2 (red) ws determined for three iologicl repets in LAMP2 sc cts B dc cts B pro-cts L sc/dc cts L Fluorescence units Cthepsin L 1 d lc 1 d inv Lysosoml Cytosolic t lest focl plnes per section. A shrp decrese is pprent in involution time points ( P <.5, P <.1 s determined y Student s t -test, n = 3). (e) Increse in cytosolic cthepsin B nd L ctivity ws mesured fter sucellulr frctiontion of mmmry glnd smples from lcttion nd involution time points. The dt re the verge of three iologicl repets. All vlues re mens±s.d. (f) Incresed lekiness of purified mmmry lysosomes from 1 dy involution time point, compred with 1 dy lcttion time point, shown y incution of the lysosomes in neutrl uffer t C for the indicted time. After re-pelleting, the mounts of cthepsins B nd L retined in the lysosomes s well s the mounts leked into the superntnt, were ssessed y immunolotting, with LAMP2 s lysosoml mrker. All of the ove were crried out with t lest three independent iologicl smples. All vlues re mens±s.d. Uncropped imges of lots re shown in Supplementry Fig. S9. With the onset of involution, we oserved shrp, LMP-medited decline in lysosoml cthepsin B ctivity in ll mice studied (Figs 3e nd 4d). Although lysosoml lekiness nd concomitnt loss of lysosoml cthepsin B ctivity is unchnged in the Stt3-knockout glnds, corresponding increse in cytosolic cthepsin ctivity ws not pprent (Fig. 4d, compre lnes.5 nd 1 dy inv in left nd right pnels). This implies tht Stt3 does not ffect the lekiness of the lysosomes per se, ut rther diminishes cytosolic cthepsin ctivity y some mechnism in ddition to trnscriptionl regultion of cthepsin gene expression. Serpins re lrge clss of suicide protese inhiitors. Memers of this fmily tht re specific for cysteine cthepsins, such s Spi2A in mice nd SRP-6 in Cenorhditis elegns, hve een shown to protect ginst cthepsin-medited cell deth in memory T cells nd fter toxic insults respectively 6,3. Anlysis of microrry dt from mmmry developmentl time course reveled tht mrna for Spi2A (encoded y Serpin3g; ref. 31) ws highly induced during lcttion 32. Spi2A mrna expression ws mximl t dy 1 lcttion, t the time when LAMP2 ws downregulted, nd dropped precipitously with the onset of involution, wheres clde B serpins hd inverse expression profiles (Figs 3 nd 5 nd Supplementry Fig. S7). This is congruent with its function s cthepsin inhiitor nd sfegurd mechnism ginst LM- PCD. Strikingly, the Stt3-knockout mice show strong trnscriptionl upregultion of Spi2A (up to -fold) throughout involution, thus explining the lck of cytosolic cthepsin ctivity mesured in Stt3- deficient mmmry tissue (Figs 5 nd 4d,e). Interestingly, lthough the upstrem flnking Serpin3f is regulted in similr mnner, this is not the cse for the downstrem flnking Serpin3h, rguing ginst generlized ctivtion of the serpin locus (Supplementry Fig. S7,c). 36 NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

5 L E T T E R S f g M r (k) d lc d involution C KO C KO C KO C KO C KO C KO pstt3 Dys OSM pstt3 tstt3 sc cts B sc cts L dc cts L β-ctin M r (k) Stt3 control OSM cthepsin B / Hoechst Stt3 knockout 3 d involution h tstt3 pro-cts B long exp. pro-cts B sc cts B pro-cts L sc cts L dc cts L β-ctin c d e Reltive expression Figure 4 Stt3 regultes cthepsin B nd L expression. () Dely of mmmry glnd involution in Stt3-knockout mice, compred with Stt3 control mice, shown y hemtoxylin- nd eosin-stined sections (scle r, µm). () Reduced mounts of totl nd tyrosine-phosphorylted Stt3 (tstt3; pstt3) nd cthepsins B nd L (cts B, L; sc, single chin, dc, hevy chin of the doule-chin form) in Stt3-knockout (KO), when compred with control (C), mice shown y western lotting. (c) Reduced expression of cthepsin nd cthepsin l in Stt3-knockout mice mesured y quntittive rel-time PCR reltive to cyclophilin. (d,e) Reduced lysosoml (fter 2 dys involution) nd cytosolic (throughout involution) ctivity of cthepsins B nd L in Stt3-knockout mice when compred with Stt3 controls, shown y sucellulr frctiontion nd susequent cthepsin ctivity mesurement. All vlues ove re mens±s.d. for three independent mesurements nd re representtive of three independent iologicl repets. (f) The mmmry epithelil cell line EpH4 ws stimulted with OSM ( ng ml 1 ) or crrier (.1% BSA in PBS) for the indicted time. Incresed tyrosine-phosphoryltion of Stt3 Reltive expression Fluorescence units Fluorescence units d lc Cthepsin L lysosoml Stt3 knockout i Lysotrcker 2.5 Cts B exp j 7 * OSM R OSM FL2 3 Cycloheximide Cell counts Cthepsin B Stt3 knockout.5 d inv 1 d inv 2 d inv 3 d inv Cthepsin B lysosoml Stt3 knockout 4 d inv 1 d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv 1 d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv Reltive expression Fluorescence units Fluorescence units d lc.5 d inv Cthepsin L 1 d inv 2 d inv 3 d inv Cthepsin B cytosolic Stt3 knockout Stt3 knockout *** 4 d inv 1 d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv Cthepsin L cytosolic Stt3 knockout 1 d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv Percentge of cells in R1 + OSM nd concomitnt increse of cthepsins B nd L re shown y western lot. (g) EpH4 cells were stimulted s in f for 3 dys. Cells were susequently fixed nd stined for cthepsin B (green). The cells were nlysed y deconvolution microscopy nd show redistriution of cthepsin B from perinucler locliztion to diffused pttern throughout the cytosol (scle r, µm). (h) EpH4 cells were co-stimulted with OSM ( ng ml 1 ) nd cycloheximide (1 µg ml 1 ) for 6 h nd the mrna levels for cthepsin were nlysed y quntittive rel-time PCR. An increse in cthepsin mrna is pprent fter OSM stimultion. Vlues re mens ± s.d. from three independent iologicl repets ( P <.5, s determined y Student s t -test). (i) Representtive plot of EpH4 cells treted s in f for 3 dys nd susequently stined with the lysosomotropic dye Lysotrcker Red (Invitrogen, nm). OSM-treted cells showed popultion of reduced stining, resulting from LMP. (j) Quntifiction of three independent experiments s descried in i ( P <.1, s determined y Student s t -test; vlues re mens ± s.d.). Uncropped imges of lots re shown in Supplementry Fig. S9. To mimic the inhiition of cthepsin ctivity y high Spi2A levels in the Stt3-knockout mice, we injected Stt3 control mice with the cell permele cthepsin B inhiitor CA-74Me. After three dys of tretment, we oserved sustntil dely in the regression of the mmmry glnd, compred with vehicle-injected controls. Notly, the extent of cthepsin B inhiition correlted strongly with the dely in regression, demonstrting crucil role for cthepsin B in involution. (r =.97, Fig. 5c,d.) Finlly, using EpH4 mmmry epithelil cells, we could effectively induce cell deth y stimultion with OSM to ctivte Stt3, s occurs in NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

6 Percentge of re occupied y dipocytes Percentge of residul cthepsin B ctivity L E T T E R S c (% ctivity) In 1 (46% ctivity) d Reltive expression Dys lcttion Dys involution % dipocytes % residul cts B ct r = Co 1 Co 2 Co 3 In 1 In 2 In 3 Vehicle injected CA-74 Me injected 3 dys involution g OSM DMSO + S3I-1 + M r (K) pstt3 tstt3 sc cts B β-ctin pro-cts L sc cts L dc cts L p55α pα h Reltive expression e Percentge of ded cells 2, 2, 1, 1, Propidium iodide Stt3 knockout 1 d lc Dys involution n.s. OSM + + DMSO + + S3I Figure 5 Inhiition of cytosolic cthepsin ctivity prevents Stt3-medited cell deth. () Expression of spi2 is high in lcttion nd drops in involution, s shown y quntittive rel-time PCR reltive to cyclophilin expression in wild-type mice. () Expression of spi2 further increses during involution in Stt3-knockout mice (grphs normlized to respective smples t 1 dys lcttion; vlues in nd represent mens±s.d. for three independent mesurements nd re representtive of three independent iologicl smples). (c,d) Mice were dministered 4 mg kg 1 of the cthepsin B inhiitor CA-74Me (In, inhiitor-injected mice). (c) Representtive microgrphs showing the dely in involution (scle r, µm). (d) Delyed involution on cthepsin B inhiition is pprent when quntifying the re occupied y dipocytes. The dely correltes with the extent of inhiition s expressed y the Person coefficient (r =.97). The vlues represent individul mice. Vlues of white rs represent mens ± s.d. (e,f) Tretment of EpH4 cells with OSM ( ng ml 1 ) for 72 h efficiently induces cell deth, which cn e rogted y the cthepsin B inhiitor CA-74Me (1 µm). 1 3 OSM Annexin V i Initite (involution) LMP TNFα TNF α? ROS? TNF α Clpins? receptor Clpins ROS In 2 (4% ctivity) OSM/CA-74Me In 3 (% ctivity) 1 OSM CA-74Me LMP Cthepsins Cthepsins Pro-cthepsins Lysosome Cthepsins f Percentge of ded cells Sensitize (lcttion) LAMP1/ Cthepsins Cells undergoing PCD n.s. Stt3 Spi Execute (involution) pstt3 LIF/OSM Cthepsins Spi2A LIFR/ gp13 Cell deth ws ssessed with Annexin V/propidium iodide co-stining nd flow cytometry. (e) Representtive dot plots for ech tretment; (f) quntifiction of totl cell deth in three independent experiments. Vlues represent mens ± s.d. of three independent iologicl repets. There ws no significnt (n.s.) difference etween control smples nd smples treted with OSM nd CA-74Me s determined y Student s t -test. (g) EpH4 cells were stimulted with OSM ( ng ml 1 ), wheres Stt3 ws inhiited with the smll-molecule inhiitor S3I-1 ( µm). Immunolotting confirmed reduced phosphorylted Stt3 on inhiitor tretment, s well s reduction of cthepsins B nd L nd the estlished Stt3 trget pα/p55α. (h) Cell deth ws mesured s in e with inhiition of Stt3 with S3I-1 ( µm). Cell deth ws reduced to levels similr to those seen with the inhiitor lone, showing protective effect of Stt3 inhiition. All vlues represent mens±s.d. for minimum of three independent experiments. Uncropped imges of lots re shown in Supplementry Fig. S9. (i) Proposed model of the findings, in which Stt3 is needed for the execution of LM-PCD. Further detils in the text. involution. Arogtion of cthepsin B ctivity with CA-74Me reduced cell deth to ckground levels, wheres the pn-cspse inhiitor z-vad-fmk showed lmost no effect (Fig. 5e,f nd Supplementry Fig. S8). Moreover tretment of OSM-stimulted EpH4 cells with smll-molecule Stt3 inhiitor (S3I-1) decresed protein levels of cthepsins B nd L nd reduced cell deth significntly (Fig. 5g,h). Thus, we could recpitulte our in vivo findings in mmmry epithelil cells in culture. We hve previously shown tht the NF-κB pthwy is ctivted during mmmry glnd involution, nd Spi2A is known trget of NF-κB in memory T cells 8,33. As Stt3 cn lock the trnscription of NF-κB trgets y inding to, nd inhiiting, p65/p suunits 34, we propose tht rogting Stt3 signlling in Stt3-knockout mice will olish the inhiitory effect of Stt3 on the NF-κB pthwy, promoting strong overexpression of Spi2A. This would give Stt3 n importnt role in modulting NF-κB signlling, fvouring cell deth rther thn survivl signture. Tken together, we hve shown tht during involution of the mmmry glnd, epithelil cells undergo lysosoml-dependent PCD, 38 NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

7 L E T T E R S which is independent of executioner cspses. To our knowledge this is the first time tht this cell deth mechnism hs een descried under physiologicl conditions, which clls for ressessment of other cell deth events in which cspses might e ctivted not s cuse, ut s consequence, of PCD. Our dt indicte multistep process leding to LM-PCD (Fig. 5i). In the first stge, during lcttion, the cells my ecome sensitized to LMP y the downregultion of the lysosoml memrne proteins LAMP1 nd 2. This is followed y the initition of LMP, y signls tht hve yet to e defined. However, TNFα signlling nd elevted C 2+ levels, oth occurring during erly involution, hve een shown to induce LMP (refs 2,35,36). The third step in LM-PCD cn e termed the execution of LMP, ecuse LMP lone does not seem to kill cells efficiently. This is ccomplished y Stt3 in t lest two wys: (1) y upregulting levels of cthepsins B nd L, which re known to ct s executioner proteses when in the cytosol, nd (2) y downregulting trnscription of the endogenous cysteine cthepsin inhiitor Spi2A. Our findings show mechnism for Stt3-medited cell deth nd revel direct connection etween Stt3, cysteine cthepsins nd LM-PCD. Prdoxiclly, Stt3 is constitutively ctivted in numer of different cncers nd elevted levels of cthepsin B hve een shown to promote metstsis 1,. Elucidtion of how tumour cells with chronic ctivtion of Stt3 evde this type of cell deth is likely to revel new, more directed, trgets for therpeutic purposes. METHODS Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry Informtion is ville on the Nture Cell Biology wesite ACKNOWLEDGEMENTS We thnk J. Skepper for help with the electron microscopy nd B. Potter for histologicl preprtions. We thnk lso P. Cme for the TUNEL dt nd W. Khled for discussions. This work hs een supported y PhD studentship from the Pthology Deprtment, University of Cmridge (P.A.K.), Brest Cncer Cmpign PhD studentship (A.D.S. nd W.L.), Itlin Cncer Reserch Assocition (V.P.) nd Biotechnology nd Biologicl Sciences Reserch Council grnt no. BB/C6836/1 (R.W.E.C. nd N.O.). AUTHOR CONTRIBUTIONS P.A.K. crried out project design nd ll experiments, except those stted seprtely nd wrote the mnuscript; A.D.S. designed nd crried out the in vitro studies; W.L. crried out studies on cspse-knockout mice; N.O. nd R.W.E.C. generted the p35 trnsgenic mouse nd provided dt; B.K. crried out cspse studies; J.T. provided Stt3 inhiitor; V.P. provided Stt3 fl/fl mice; R.A.F. provided ll cspse-knockout mice; C.J.W. crried out project design nd wrote the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Kroemer, G. & Jttel, M. Lysosomes nd utophgy in cell deth control. Nt. Rev. Cncer 5, (5). 2. Guiccirdi, M. E., Leist, M. & Gores, G. J. Lysosomes in cell deth. Oncogene 23, (4). 3. Hitomi, J. et l. Identifiction of moleculr signling network tht regultes cellulr necrotic cell deth pthwy. Cell 135, (8). 4. Lockshin, R. A. & Zkeri, Z. Cspse-independent cell deth? Oncogene 23, (4). 5. Nylndsted, J. et l. Erdiction of gliolstom, nd rest nd colon crcinom xenogrfts y Hsp7 depletion. Cncer Res. 62, (2). 6. Luke, C. J. et l. An intrcellulr serpin regultes necrosis y inhiiting the induction nd sequele of lysosoml injury. Cell 13, (7). 7. Guiccirdi, M. E., Miyoshi, H., Bronk, S. F. & Gores, G. J. Cthepsin B knockout mice re resistnt to tumor necrosis fctor-α-medited heptocyte poptosis nd liver injury: implictions for therpeutic pplictions. Am. J. Pthol. 9, (1). 8. Liu, N. et l. NF-κB protects from the lysosoml pthwy of cell deth. EMBO J. 22, (3). 9. Grivennikov, S. et l. IL-6 nd Stt3 re required for survivl of intestinl epithelil cells nd development of colitis-ssocited cncer. Cncer Cell, (9). 1. Vsiljev, O. et l. Reduced tumour cell prolifertion nd delyed development of high-grde mmmry crcinoms in cthepsin B-deficient mice. Oncogene 27, (8). 11. Foghsgrd, L. et l. Cthepsin B cts s dominnt execution protese in tumor cell poptosis induced y tumor necrosis fctor. J. Cell Biol. 3, (1). 12. Holler, N. et l. Fs triggers n lterntive, cspse-8-independent cell deth pthwy using the kinse RIP s effector molecule. Nt. Immunol. 1, (). 13. Kgedl, K., Zho, M., Svensson, I. & Brunk, U. T. Sphingosine-induced poptosis is dependent on lysosoml proteses. Biochem. J. 359, (1). 14. Nkym, M. et l. Multiple pthwys of TWEAK-induced cell deth. J. Immunol. 168, (2).. Ymshim, T. Impliction of cysteine proteses clpin, cthepsin nd cspse in ischemic neuronl deth of primtes. Prog. Neuroiol. 62, (). 16. Lscelles, A. K. & Lee, C. S. Lcttion: A Comprehensive Tretise Vol. 4. (Acdemic, 1978). 17. Nguyen, A. V. & Pollrd, J. W. Trnsforming growth fctor β3 induces cell deth during the first stge of mmmry glnd involution. Development 127, (). 18. Kritikou, E. A. et l. A dul, non-redundnt, role for LIF s regultor of development nd STAT3-medited cell deth in mmmry glnd. Development 13, (3). 19. Chpmn, R. S. et l. Suppression of epithelil poptosis nd delyed mmmry glnd involution in mice with conditionl knockout of Stt3. Genes Dev. 13, (1999).. Lund, L. R. et l. Two distinct phses of poptosis in mmmry glnd involution: proteinse-independent nd -dependent pthwys. Development 122, (1996). 21. Thngrju, M. et l. C/EBP δ is crucil regultor of pro-poptotic gene expression during mmmry glnd involution. Development 132, (5). 22. Bxter, F. O., Neoh, K. & Tevendle, M. C. The eginning of the end: deth signling in erly involution. J. Mmm. Glnd Biol. Neoplsi 12, 3 13 (7). 23. Overholtzer, M. et l. A nonpoptotic cell deth process, entosis, tht occurs y cell-in-cell invsion. Cell 131, (7). 24. Mrti, A. et l. Mouse mmmry glnd involution is ssocited with cytochrome c relese nd cspse ctivtion. Mech. Dev. 14, (1).. Zhou, Q. et l. Interction of the culovirus nti-poptotic protein p35 with cspses. Specificity, kinetics, nd chrcteriztion of the cspse/p35 complex. Biochemistry, (1998). 26. Fehrencher, N. et l. Sensitiztion to the lysosoml cell deth pthwy y oncogene-induced down-regultion of lysosome-ssocited memrne proteins 1 nd 2. Cncer Res. 68, (8). 27. Zrgoz, R. et l. Nitrtion of cthepsin D enhnces its proteolytic ctivity during mmmry glnd remodelling fter lcttion. Biochem. J. 419, (9). 28. Mohmed, M. M. & Slone, B. F. Cysteine cthepsins: multifunctionl enzymes in cncer. Nt. Rev. Cncer 6, (6). 29. Tiffen, P. G. et l. A dul role for oncosttin M signling in the differentition nd deth of mmmry epithelil cells in vivo. Mol. Endocrinol. 22, (8). 3. Liu, N. et l. Serine protese inhiitor 2A is protective fctor for memory T cell development. Nt. Immunol. 5, (4). 31. Inglis, J. D. & Hill, R. E. The murine Spi-2 proteinse inhiitor locus: multigene fmily with hypervrile rective site domin. EMBO J. 1, (1991). 32. Clrkson, R. W., Wylnd, M. T., Lee, J., Freemn, T. & Wtson, C. J. Gene expression profiling of mmmry glnd development revels puttive roles for deth receptors nd immune meditors in post-lcttionl regression. Brest Cncer Res. 6, R92 R19 (4). 33. Clrkson, R. W. et l. NF-κB inhiits poptosis in murine mmmry epitheli. J. Biol. Chem. 2, (). 34. Yu, Z., Zhng, W. & Kone, B. C. Signl trnsducers nd ctivtors of trnscription 3 (STAT3) inhiits trnscription of the inducile nitric oxide synthse gene y intercting with nucler fctor κb. Biochem. J. 367, 97 (2). 35. Bxter, F. O. et l. IKKβ/2 induces TWEAK nd poptosis in mmmry epithelil cells. Development 133, (6). 36. Reinhrdt, T. A. & Lippolis, J. D. Mmmry glnd involution is ssocited with rpid down regultion of mjor mmmry C2+-ATPses. Biochem. Biophys. Res. Commun. 8, (9).. Yu, H., Prdoll, D. & Jove, R. STATs in cncer inflmmtion nd immunity: leding role for STAT3. Nt. Rev. Cncer 9, (9). NATURE CELL BIOLOGY VOLUME 13 NUMBER 3 MARCH Mcmilln Pulishers Limited. All rights reserved.

8 M E T H O D S DOI: 1.138/nc2171 METHODS Animl husndry. Mice crrying the stt3 gene flnked y loxp sites 38 were crossed with strin contining β-lctogloulin-promoter-driven Cre gene 39 (oth on mixed ckground). Cspse 3, 6 nd 7 mice 4 42 re on C57BL/6 ckground. Mice with doxycycline-inducile p35 trnsgene re on the FVB ckground. Trnsgenic induction ws chieved with the dministrtion of 2 mg ml 1 doxycycline (Sigm) in the drinking wter for 7 dys ccompnied y intrperitonel injection (5 mg kg 1 ) for 5 dys efore collection. C57BL/6 mice were purchsed from Hrln ls. The mice were red in regulr cges with food nd wter d liitum. Virgin femle mice, 8 14 weeks old, were mted nd mles were susequently removed efore irth to void second pregnncies. Dms were killed t indicted time points. For involution studies, pups were removed t 1 dys of lcttion. At lest three mice were used for ech time point in every experiment. All nimls were treted ccording to locl ethicl committee nd the UK Home Office guidelines nd killed through CO 2 or disloction of the neck. Cthepsin B inhiition in vivo. Stt3 control mice were mted s descried ove nd pup numers were normlized to eight. At dy 1 of lcttion, the pups were removed nd the mice were dministered 4 mg kg 1 CA-74Me (Peptnov, 5 mg dissolved in 8 µl dimethylsulphoxide nd 1,5 µl PBS) y intrperitonel injection every 12 h. mice received vehicle lone. After 3 dys the mice were euthnized nd the glnds were fixed nd nlysed. Assessment of delyed involution in vivo. To quntify the dely of mmmry glnd involution following cthepsin B inhiition, the re occupied y dipocytes ws ssessed on hemtoxylin- nd eosin-stined mmmry glnd sections using ImgeJ softwre. The men ctivity of the control mice ws set s % nd the mounts of residul cthepsin B ctivity were relted to this vlue. The correltion ws tested with the Person coefficient. Trnsmission electron microscopy. Trnsmission electron microscopy ws crried out s previously descried 43. Hemtoxylin nd eosin stining. Hemtoxylin nd eosin stining ws crried out s previously descried 44. TUNEL stining of mmmry glnd sections. The rection ws crried out on fixed tissues using the ApoTg indirect fluorescence stining kit (S711, Chemicon) following the mnufcturer s instructions. Sucellulr frctiontion. Lymph node divested numer four glnds were homogenized (pproximtely strokes) in tight-fitting hndheld homogenizer in 1 ml of sucellulr frctiontion uffer (HEPES-KOH mm, sucrose 2 mm, KCl 1 mm, MgCl mm, EDTA 1 mm, EGTA 1 mm, dithiothreitol 8 mm, Pefloc 1 mm (Fluk), t ph 7.5). Deris nd nuclei were pelleted t 7g (3, r.p.m., BeckmnCoulter Optim L- XP Ultrcentrifuge, 4 C) for 12 min. The superntnt ws spun t 1,g (12,9 r.p.m.) for 35 min to pellet lysosomes nd other orgnelles. The pellet ws rinsed nd collected in 3 ml sucellulr frctiontion uffer s lysosoml frction. The superntnt, contining cytosolic nd extrcellulr components, ws clered of microsomes y n extr spin t,g (4, r.p.m.) for 1 h nd collected s cytosolic frction. Orgnelles were disrupted y three cycles of freezing nd thwing. Lysosoml-lekiness ssy. Lysosomes from two whole mmmry glnds excluding lymph nodes were prepred s descried, with the exception tht the sucellulr frctiontion uffer ws mde up with Complete protese inhiitor (Roche) insted of Pefloc (sucellulr frctiontion protese inhiitor). They were wshed once in sucellulr frctiontion uffer nd re-pelleted in tle-top centrifuge (Hereus Fresco-17 centrifuge, 4 C, min, 12, r.pm., 13,8g). After resuspending in 4 µl sucellulr frctiontion protese inhiitor uffer, equl volumes ( µl) were either pelleted immeditely or incuted for the indicted time t C under gentle gittion. Susequently, the lysosomes were re-pelleted s ove. The superntnt ws removed nd snp frozen. The pellet ws resuspended in 1 µl RIPA uffer ( mm Tris, 1% NP4,.% sodium deoxycholte, 1 mm NCl, 1 mm EGTA nd Complete protese inhiitor), nd orgnelles were lysed on ice for 3 min with intermittent vortexing. Memrnes were pelleted in tle-top centrifuge (Hereus Fresco-17 centrifuge, 4 C, min, 12, r.p.m., 13,8g), the superntnt ws removed nd equl mounts of the resuspended pellet nd the superntnt were nlysed y western lot. Cthepsin B nd L ctivity. For whole cell cthepsin ctivity, lymph node divested numer four glnds were snp frozen nd ground to fine powder in mortr. Totl protein ws extrcted with modified RIPA uffer ( mm Tris, 1% NP4,.% sodium deoxycholte, 1 mm NCl, 1 mm EGTA nd Pefloc 1 mm (Fluk), t ph 7.4). To mesure lysosoml nd cytosolic cthepsin ctivity, sucellulr frctiontion ws crried out s descried erlier. In oth cses, protein levels were ssessed with the BCA Protein Assy (Thermo Scientific) nd equl mounts of protein (4 µg) were dded to totl of µl cthepsin rection uffer (sodium cette mm, EDTA 8 mm, dithiothreitol 8 mm nd Pefloc sucellulr frctiontion uffer 1 mm, t ph6). Cthepsin B + L ctivity ws mesured fter incution ( min, C) with the fluorescent sustrte Z- Phe-Arg-AMC (Cliochem, µm) in Synergy HT Multi-Detection Microplte Reder (Bio-TEK; excittion 38 nm, emission 442 nm). In prllel, smple with dded cthepsin B inhiitor CA-74 (Peptnov; 5 µm) ws mesured to determine cthepsin L ctivity. The difference etween redings corresponds to the cthepsin B ctivity. Cspse 3, 6 nd 7 ctivity. Cspse ctivity ws mesured with the fluorescent sustrtes AC-DEVD-AMC for cspses 3 nd 7 nd AC-VEID-AMC for cspse 6. Protein ( µg) ws diluted in cspse rection uffer (1 mm HEPES t ph 7.5, mm NCl nd 8 mm dithiothreitol), nd incuted with finl concentrtion of µm of the respective sustrte. The smples were incuted t C for 5 h nd fluorescence ws mesured in Synergy HT Multi-Detection Microplte Reder (Bio-TEK; excittion 38 nm, emission 442 nm). Quntittive rel-time PCR. RNA extrction, complementry DNA synthesis nd quntittive rel-time PCR were crried out s previously descried 44. Expression levels were mesured for cyclophilin (housekeeping gene), cthepsin, cthepsin l nd spi2 using the following primers: cyclophilin A forwrd, 5 -CCT TGG GCC GCG TCT CCT T-3, reverse, 5 -CAC CCT GGC ACA TGA ATC CTG-3 ; cthepsin B forwrd, 5 -TCC TTG ATCC TTC TTT CTT GCC-3, reverse, 5 -ACA GTG CCA CAC AGC TTC TTC-3 ; cthepsin L forwrd, 5 -ATC AAA CCT TTA GTG CAG AGT G-3, reverse, 5 -CTG TAT TCC CCG TTG TGT AGC-3 ; Spi2A forwrd, 5 -TTT CCA GCA ACC TCT CAA GGC-3, reverse, 5 -CTG GGT GTG ATT GCC CAC ATA-3. Primers were designed using the PrimerBnk wesite ( Immunolotting. Smple preprtion nd immunolotting ws crried out s previously descried 44. The following rit ntiodies from Cell Signling Technologies were used: nti-cleved cspse 3 (9661), nti-cleved cspse 6 (9761), nti-cleved cspse 7 (9491), nti-cspse 3 (9665), nti-cspse 6 (9762), nticspse 7 (9492) nd nti-phospho-stt3 (Tyr75: 9131). The following ntiodies from Acm were used: rit nti-cthepsin B (33538), rt nti-lamp2 (13524) nd rit nti-β-ctin (8227). Other commercil ntiodies used were: rt nti-cthepsin L (R&D Systems, MAB9521), mouse nti-stt3 (BD Trnsduction Lortories, 6119) nd rit nti-pn-p85 (Millipore, , lso detects pα/p55α suunits). All ntiodies were used t stndrd dilution of 1:1,. Secondry horserdish peroxidse (HRP)-conjugted ntiodies were purchsed from Dko. Immunohistochemistry. Tissue sections were prepred s previously descried 44. For ssessment of cthepsin locliztion on tissue sections, the ckground ws reduced y incuting the sections in.1 M glycine in PBS (ph 7.4) for min. Susequently, the tissue ws permeilized with.1% sponin (Sigm) in PBS nd locked in 1% norml got serum (Dko) in PBS,.1% sponin. Primry ntiodies used were: rit nti-cthepsin B (Acm, 1:), rt nti-lamp2 (Acm 13524, 1:), nd for the cspses 3, 6 nd 7, cc3, #9661; cc6, #9761; cc7, #9491; ll rit, ll Cell Signling Technologies nd ll t 1: dilution. Secondry ntiodies used were Alex Fluor 488 got-nti-rit-igg (Invitrogen, 1:) nd Cy3 got-nti-rt-igg (Invitrogen, 1:). Nuclei were counterstined with Hoechst (Sigm, 1:1,). The pictures were cquired on Zeiss Axiopln 2 microscope with the ApoTome set-up for deconvolution microscopy. Mnder s coefficient ws determined using the ImgeJ plug-in Mnder coefficient y T. Collins. It ws crried out on t lest four z stcks with t lest cross-sections for every time point nlysed. For tissue-culture smples, EpH4 cells were seeded on chmer slides (NUNC). Cells were fixed with 4% prformldehyde, permeilized with 1% sponin in PBS nd locked in 1% norml got serum (Dko) in PBS,.1% sponin. Cell culture. EpH4 cells were seeded on plstic or chmer slides nd grown in DMEM (Gico) contining 1% FCS (Sigm). At % confluency, cells were stimulted with finl concentrtion of ng ml 1 recominnt mouse Oncosttin M (495-MO, R&D Systems) or crrier (.1% BSA in PBS). Medium ws renewed every 48 h. For Stt3 inhiition, the smll-molecule inhiitor S3I-1 ( µm) ws dded longside OSM. Tretment of cells with cycloheximide. EpH4 cells were seeded on cm 2 dishes (4, cells) in DMEM/1% FCS. The following dy, cells were stimulted for 6 h with OSM ( ng ml 1 ) nd cycloheximide (Cliochem, 1 µg ml 1 ). Corresponding mounts of crriers (PBS with.1% BSA) were used s control 11 Mcmilln Pulishers Limited. All rights reserved. NATURE CELL BIOLOGY

9 DOI: 1.138/nc2171 M E T H O D S tretments. After 6 h, the medium ws spirted, cells were collected in 1 ml of Trizol (Invitrogen) nd mrna ws extrcted nd quntified. Quntifiction of cell deth y flow cytometry. EpH4 cells were seeded on six-well Nunclon Surfce pltes (Nunc,, cells per well) in DMEM, 1% FCS (Gico/Sigm). Stimuli (OSM, R&D, ng ml 1 ; CA-74Me, Sigm, 1 µm; S3I-1, µm) were dded in DMEM, 1% FCS, the following dy nd renewed fter 48 h. Cells were collected nd resuspended in µl Annexin V Binding Buffer (1 mm HEPES/NOH t ph 7.4, 14 mm NCl nd 2.5 mm CCl 2 ) nd susequently stined for 1 min in the drk with 5 µl of Annexin V-FITC (Gico). Next, propidium iodide (Sigm, 1 ng ml 1 ) ws dded for 5 min. Cells were wshed with Annexin V Binding Buffer, nd resuspended in µl of Annexin V Binding Buffer. Two-colour flow cytometry ws crried out on CyAn ADP flow cytometer (DkoCytomtion). Dt nlysis ws crried out using the FlowJo softwre (Tree Str). Lysotrcker Red stining. Cells were seeded, grown nd stimulted s descried for the cell deth ssy. Susequently, the cells were collected, resuspended in 1 ml culture medium nd incuted for 5 min t C. LysoTrcker Red DND-99 (Invitrogen, nm) ws dded to the suspension nd incuted t C in the drk for 3 min. Single-colour flow cytometry ws crried out on CyAn ADP flow cytometer (DkoCytomtion), nd the dt were nlysed using the Summit 4.3 softwre (DkoCytomtion). Sttisticl nlysis. Every experiment ws crried out with t lest three independent iologicl smples. Where pproprite, sttisticl significnce ws ssessed with Student s t-test. The correltion etween cthepsin B inhiition nd delyed involution ws ssessed with the Person coefficient using Microsoft Excel. 38. Alonzi, T. et l. Essentil role of STAT3 in the control of the cute-phse response s reveled y inducile gene inctivtion [correction of ctivtion] in the liver. Mol. Cell Biol. 21, (1). 39. Selert, S. et l. Efficient BLG-Cre medited gene deletion in the mmmry glnd. Trnsgenic Res. 7, (1998). 4. Kuid, K. et l. Decresed poptosis in the rin nd premture lethlity in CPP32-deficient mice. Nture 384, (1996). 41. Lkhni, S. A. et l. Cspses 3 nd 7: key meditors of mitochondril events of poptosis. Science 311, (6). 42. Zheng, T. S. et l. Deficiency in cspse-9 or cspse-3 induces compenstory cspse ctivtion. Nture Med. 6, (). 43. Wrley, A., Powell, J. M. & Skepper, J. N. Cpillry surfce re is reduced nd tissue thickness from cpillries to myocytes is incresed in the left ventricle of streptozotocin-dietic rts. Dietologi 38, (1995). 44. Khled, W. T. et l. The IL-4/IL-13/Stt6 signlling pthwy promotes luminl mmmry epithelil cell development. Development 134, (7). NATURE CELL BIOLOGY 11 Mcmilln Pulishers Limited. All rights reserved.

10 DOI: 1.138/nc min DNse 3 min DNse 1 d inv 1 d inv TUNEL/Hoechst Figure S1 Hyper-compcted nuclei of shed cells remin TUNEL negtive even fter DNse I tretment. As positive control for the TUNEL stining, tissue sections were pre-treted with DNse I (Sigm). To this end, the slides were incuted in DN uffer (3mM Trizm Bse, 4mM MgCl 2,,1 mm DTT, ph7.2) for 5 min. prior to incution with DNse I (5, U/ml in DN) for indicted times. DNse I ws susequently removed through 3 wshes with dh 2 O. The nuclei of shed cells undergoing PCD remin TUNEL negtive even fter prolonged tretment with DNse I, while most of the nuclei of vile cells ecome TUNEL positive. This underlines the hyper-compcted structure tht these nuclei cquire, s the TUNEL regents cnnot penetrte the nucleus. Scle rs: µm Mcmilln Pulishers Limited. All rights reserved.

11 % re occupied y dipocytes 1 5 Progression of involution in C3+6 KO mice C3+6 KO 1 d inv 2 d inv 3 d inv qrt-pcr for P35 expression Reltive p35 expression; +Dox/-Dox 1 1 Figure S2 Cspses re not required for involution., Mesurement of the re occupied y dipocytes is good indictor to ssess the kinetics of involution. The re ws mesured using the imging softwre ImgeJ on t lest five high power visul fields nd shows tht there is no significnt difference etween the cspse 3 nd 6 doule KO mice nd control nimls. For every time point three independent histologicl sections from three iologicl repets were quntified. Vlues represent mens ± s.d., mrna ws hrvested from mmmry glnds of i-trnsgenic (rtta/p35) mice treted with systemic Doxycycline or untreted controls. Reltive differences in p35 gene expression etween pired (littermte) smples were clculted y using the 2 -ΔΔCT method, y normlising the CT vlues for ech smple to the CT vlues for the et-ctin housekeeping gene. The verge expression of p35 in control mice ws set to 1 nd the lck dots represent fold increse of the p35 mrna in ech of three mice. The horizontl r represents the verge of these vlues. An verge of 6 fold difference in p35 expression ws oserved Mcmilln Pulishers Limited. All rights reserved.

12 verge signl intesity Lmp1 Lmp2 virgin dys gesttion dys lcttion dys involution Figure S3 LAMP1 nd LAMP2 re trnscriptionlly down regulted during lcttion. Microrry nlysis of 12 time point developmentl time course of the mmmry glnd 31. LAMP1 nd LAMP2 re oth trnscriptionlly down-regulted during lcttion Mcmilln Pulishers Limited. All rights reserved.

13 Fluoresc ence U nits Totl cthepsin ctivity CA-74Me Z-FF-FMK d lc 1 d inv 2 d inv 3 d inv T L C T L C T L C T L C Tulin LAMP2 sc cts B dc cts B c % crosscontmintion Actin LAMP2 Cth B Figure S4 ssys for cthepsin ctivity mesurement nd tissue frctiontion., The cthepsin like ctivity of tissue lystes from 2 dy involution time points ws mesured with the ddition of the cthepsin B inhiitor CA-74, the cthepsin L nd prtil cthepsin B inhiitor Z-FF- FMK (µm, Cliochem #219421) or oth. Inhiition of oth cthepsins left lmost no residul cthepsin ctivity, demonstrting tht the fluorescent sustrte Z-Phe-Arg-AMC is lmost exclusively cleved y these cthepsins in our system (n=3, vlues represent mens ± s.d.)., The qulity of the sucellulr frctiontion ws ssessed vi western lot using specific mrkers for the cytosolic (tuulin, rt, ACm #616) nd the lysosoml (LAMP2) frction (T=totl lyste, L=lysosoml frction, C= cytosolic frction). Furthermore the distriution of cthepsin B ws nlyzed, showing redistriution eyond levels of crosscontmintion. The western lot ws developed using the ChemiDoc imger (BioRd) nd the supplied Quntity One softwre. This test ws routinely performed nd the smples re representtive of successful frctiontion. c, The rtio of signl mesured in the frction in which the mrker is not loclized under norml conditions nd the other cellulr comprtment. (i.e. for tuulin L/C) Mcmilln Pulishers Limited. All rights reserved.

14 FU Buffer/Pellet d lc 1 d inv Cthepsin B FU Buffer/Pellet d lc 1 d inv Cthepsin L Figure S5 Activity mesurements show incresed lekiness of lysosomes from involuting mmmry glnd compred to lctting glnd. A lysosoml lekiness ssy ws performed s descried, however using SC uffer rther thn SC-PI to llow ctivity mesurements. The cthepsin ctivity ws mesured in the pellet nd the superntnt. To ccount for ctivity loss due to longer incution periods, results re depicted s rtios of ctivity in the superntnt nd the lysosoml pellet. It cn e seen tht during involution significntly higher mounts of cthepsin ctivity re mesured in the superntnt. The experiment ws crried out six times for every time point consistently showing the sme trend. The vlues stem from one representtive ssy nd the error rs show the stndrd devition from three independent repets Mcmilln Pulishers Limited. All rights reserved.

15 1 d Lc 1 d Inv 2 d Inv Stt 3 control Stt 3 KO % ft in mmmry glnd,4,35,3,,2,,1,5 Stt3 KO 1 d inv 2 d inv 3 d inv 4 d Inv Figure S6 Filed involution in the Stt3 fl/fl, BLG-Cre mice., Histologicl confirmtion of filed involution in mice with conditionl deletion of Stt3 in the mmmry glnd epithelium during lcttion. Morphologiclly Stt3 KO mice re indistinguishle from the controls during lcttion, ut show little evidence of involution for numer of dys fter wening. Scle rs µm., The filed involution ws dditionlly quntified y mesuring the re repopulted y dipocytes s descried ove. For every time point three independent histologicl sections from three iologicl repets were quntified. Vlues represent mens ± s.d Mcmilln Pulishers Limited. All rights reserved.

16 Serpin4 verge signl intesity Serpin5 Serpin6 Serpin3g virgin dys gesttion dys lcttion dys involution R eltive E xpression Stt3 KO Serpin3f qrt PCR c R eltive E xpression Stt3 KO Serpin3h qrt PCR 1d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv 1d lc.5 d inv 1 d inv 2 d inv 3 d inv 4 d inv Figure S7 Expression profile of selected serpins., Microrry nlysis of 12 time point developmentl time course of the mmmry glnd. As opposed to the Clde A serpin Serpin3g, numer of Clde B serpins show low expression levels during lcttion, while these increse in involution. Notly Serpin4 shows lmost no expression in the mmmry glnd.,c, Quntittive rel time PCR shows tht Serpin3f, flnking Serpin3g upstrem, is similrly over-expressed in the Stt3 KO mice. This is much less striking for its downstrem flnking neighour Serpin3h. This shows differentil regultion of the Clde A locus. Primers used: Serpin3f Fwd, 5 -ATC TCC AAT GTT GTC AAG GTG-3 ; Rev; 5 TGT AAC TTT TGC CAT AAA GAG-3, Serpin3h, Fwd 5 -CCA CAG GGG TCA AAT TAA TTC-3 ; Rev, 5 -CTT GGG ATT TGT AAC TTT GGC-3. Vlues represent mens ± s.d. of representtive time course nd 3 independent mice hve een studied for ech time point Mcmilln Pulishers Limited. All rights reserved.

17 % of ded cells DMSO ZVAD 5µM OSM DMSO OSM ZVAD 5µM c Fluorescente Units 2 1 Fluorescente Units cspse 3/7 cspse 6 DMSO 5 µm 1 µm µm µm ZVAD DMSO 5µM 1µM µm µm ZVAD cthepsin B cthepsin L Figure S8 Inhiition of cspses in OSM treted EpH4 cells hs only minor effect on cell deth., Cell deth ssys were crried out s descried in mterils nd methods, while cells were supplemented with the indicted mounts of the pn-cspse inhiitor Z-VAD-FMK (ZVAD, Sigm). Vlues represent mens ± s.d. of three independent iologicl repets (n=3)., c, Protese ctivity ws mesured s descried in mterils nd methods. Cspse 3 nd 6 ctivities were mesured fter overnight incution of µg t RT with the fluorescent sustrtes AC-DEVD-AMC (Sigm, µm) nd AC- VEID-AMC (Sigm, µm), respectively. Inhiition of executioner cspses hd only minor effect on cell viility. Cthepsin ctivity ws reduced y even low mounts of ZVAD, which could lso influence cell viility. Vlues represent mens ± s.d. of three independent iologicl repets (n=3) Mcmilln Pulishers Limited. All rights reserved.

18 Figure 1 c WT C3 KO d lc d inv WT C6 KO d lc d inv WT C7 KO d lc d inv Proforms ctive Figure 2 control C3+6 KO Figure 3 dys lcttion dys involution Figure 3 f Pellet Superntnt M dys lcttion dy involution Figure 4 d lc d inv C C C C C KO KO KO KO KO 4 C KO 1 Figure 4 f dys OSM Figure S9 Uncropped western lots Mcmilln Pulishers Limited. All rights reserved.

19 Figure 5g Figure S 4 1 d lc 1 d inv 2 d inv 3 d inv T L C T L C T L C T L C 1 Figure S9 continued Mcmilln Pulishers Limited. All rights reserved.