Survival ( %) a. TNFα 18hr b. Control sirna Pellino3 sirna TNFα: 18 18hr c. Control shrna Pellino3 shrna Caspase3 Actin Control d. Control shrna Pellino3 shrna *** 100 80 60 CHX 18hr 40 TNFα+ CHX 18hr f. Control shrna 0 CHX TNF + CHX 18 hour Pellino3 shrna Pellino3 β-actin Control shrna Pellino3 shrna TNFα 0 5 15 30 60 1 0 5 15 30 60 1min I Bα p-p38 P38 38 38 e. p-jnk JNK 38 38 TNFα+ CHX: 18 18hr Caspase-3 Caspase-8 Actin 18 Actin Supplementary Figure S1.:Knockdown of Pellino3 in Hela and 1321N1 astrocytoma leads to increased sensitization to TNF-induced apoptosis. (a, b) Hela cells were transfected with control or Pellino3-specific sirna and then treated with human TNF (40 ng/ml) for 18h. (a) Cells were photographed (bar μm). (b) Cell lysates were probed by immunoblotting for expression levels of, cleaved caspase 3 and b-actin. (c, d) 1321N1 astrocytoma cells were infected with lentivirus control or Pellino3-shRNA and treated in the absence (control) or presence of human TNF (40 ng/ml) and/or cycloheximide (CHX) (10 μg/ml). (c) Cells were photographed (bar μm). Immunoblot analysis demonstrating efficacy of Pellino3 protein knockdown is shown. (d) Cells were analyzed for cell viability using MTT assays (mean +/- SEM of 3 independent experiments) ***p<0.001 (two-way ANOVA). (e) Cell lysates were immunblotted for, caspase 3, caspase 8 and b-actin. and caspase 8 are indicated by arrows. (f) Cell lysates blotted for phosphorylated JNK (p-jnk),p38 (p-p38) and total I Bα, JNK and P38
Input Input sirnap3 sirnac a. b. TNFα18hr TNFα: 18 18hr Caspase-3 Actin Pellino3 TUNEL Hoechst Merge c. IP: sirnacontrol sirnapellino3 Z-VAD-FMK : + + + + + + TNFα + CHX : 0 2 5 0 2 5hr d. IP: sirnacontrol sirnapellino3 Z-VAD-FMK : + + + + + + TNFα + CHX : 0 2 5 0 2 5hr Rip1 Caspase-8 Nonspecific IgGL Rip1 Caspase-8 Supplementary Figure S2: Knockdown of Pellino3 in HEK293 cells leads to increased sensitization to TNF-induced apoptosis. (a, b) HEK293 cells were transfected with control or Pellino3-specific sirna and treated with TNF ( ng/ml) for 18 h. (a) Cell lysates were immunoblotted for, caspase 3, Pellino3 and b-actin. (b) Cells were stained by TUNEL and Hoechst and subjected to confocal microscopy. Bar μm. (c, d) HEK293 cells were transfected with control or Pellino3-specific sirna and treated with TNF ( ng/ml), CHX (10 μg/ml) and ZVAD ( mm). Cell lysates were immunoprecipitated with an (c) anti-caspase 8 or (d) anti- antibody and blotted for coprecipitated and caspase 8. The expression levels of and caspase 8 in whole cell lysates ( input ) were also assessed.
Supplementary Figure S3: Schematic representation of mutant forms of Pellino3 used in reconstitution and overexpression studies.
In vitro K63Ub 1 IB: Ub 55 IB: UbK63only: + + + + E1: + + + + E2: + + + + Pellino3smyc: + Pellino3sFmmyc: + Pellino3sEmmyc: + Supplementary Figure S4: Mutation of the RING-like domain but not the FHA domain of Pellino3 abrogates its E3 ubiquitin ligase activity. Purified recombinant forms of myc-tagged Pellino3s, FHA domain-mutated Pellino3s(P3sFHAm) or RING-like domain-mutated Pellino3s(P3sE3m) (0.5 µg) were incubated with recombinant ubiquitin (1µg) (containing a single lysine at residue 63 (K63 Ub)) in the presence of E1 (ng) and the E2 UbcH13 (400 ng) for 2 h at 37 C. The reactions were terminated by the addition of SDS-PAGE sample buffer. Samples were subjected to PAGE and subsequently to Western immunoblotting using anti-ubiquitin and -myc antibodies.
a. IP: Flag b. IP: Flag Flag P3 Flag: P3ΔC-term Flag: -: Input + + + + + Flag P3 Full-length P3 Δ C-term Input P3 Full-length Flag P3 Δ C-term P3 Flag: + + P3ΔC-term Flag: + + -: + + P3s Full-length P3s Δ C-term P3s Full-length P3s Δ C-term c. IP: Flag d. Flag IP: P3s Full-length Flag Input P3 Full-length P3 Δ C-term Input Flag P3 Flag: P3ΔC-term Flag: + Caspase-8 : + + + + P3 Full-length Flag P3 Δ C-term P3 Flag: + P3ΔC-term Flag: + + + Caspase-8 : + + P3s Full-length P3s Δ C-term Supplementary Figure S5: The FHA domain of Pellino3 is sufficient to interact with but not caspase8. HEK293 cells were co-transfected with indicated combinations of constructs encoding Flag-tagged Pellino3s or its C-terminal truncation mutant (P3 ΔC-term(1-313aa)) and (a, b) myc-tagged or (c, d) myc-tagged caspase8. Such transfections also included co-expression of the caspase inhibitor CrmA to prevent caspase induced cleavage of and cell apoptosis. Cell lysates were immunoprecipitated (IP) using anti-flag (a, c) or anti-myc (b, d) antibodies followed by immunoblotting of immunopreciptates and cell lysates (input) using indicated antibodies.
Annexin V positive(%) Annexin V positive(%) a. Pellino3 +/+ Pellino3 -/- 40 30 10 b. Caspase 3 FasL + + + + CHX + + 0 FasL FasL+CHX β-actin c. 45 40 35 30 15 10 5 0 Control Pellino3 +/+ Pellino3 -/- Etoposide d. Etoposide + + 18 Caspase 3 β-actin Supplementary Figure S6: Loss of Pellino3 has no effect on the pro-apoptotic effects of Fas or genotoxic stress. MEFs were isolated from Pellino3+/+ and Pellino3-/- embryos and treated with (a, b) cycloheximide (CHX) (10 μg/ml) and/or anti-fas antibody (2 µg/ml) for 12h or (c, d) etoposide (0 mm) for 24 hr. (a, c) Cells were analyzed by flow cytometry for percentage of cells staining positive for annexin V staining. Data represent the mean +/- SEM of 3 independent experiments. (b, d) Cell lysates were probed by immunoblotting for expression levels of, cleaved caspase 3 and β-actin.
Annexin V positive (%) mp3δcm Retrovirus Vector Retrovirus a. Control TC 10hr b. 60 40 30 vector mpellino3 Cm ns c. Pellino3 -/- MEF Caspase 3 myc TNFα+ CHX 10hr Retro Retro Vector mp3δcm 10 GFP 30 0 CHX TNF + CHX 1 2 Actin Supplementary Figure S7: The FHA domain of Pellino3 is not sufficient to inhibit the proapoptotic effects of TNF. MEFs, from Pellino3 -/- embryos, were infected with murine stem cell virus (MSCV), lacking (vector retrovirus) or containing the myc-tagged C-terminal truncation mutant of the murine Pellino3 gene (mp3δcm). Infected cells were treated in the absence (control) or presence of murine TNF (40 ng/ml) and cycloheximide (CHX) (10 μg/ml) (TC) for 10h. (a) Cells were photographed using a phase contrast microscope (bar μm) or viewed by fluorescent microscopy to detect infected cells based on expression of MSCV-encoded GFP. (b) Cells were analyzed by flow cytometry for percentage of cells staining positive for annexin V staining. Data represent the mean +/- SEM of 3 independent experiments; n.s: not significant (two-way ANOVA). (c) Cell lysates were probed by immunoblotting for expression levels of, cleaved caspase 3, myc- Pellino3,GFP and b-actin.
Fig. 1d. Control shrna Pellino3 shrna TNFα+ CHX: 0 2 4 6 0 2 4 6hr Fig. 1e. IP: Caspase 8 Control shrna Pellino3 shrna Z-VAD-FMK : + + + + + + TNFα + CHX : 0 2 5 0 2 5hr Caspase-3 Caspase-8 18 Pellino3 Caspase 8 Caspase 8 Supplementary Figure S8: Full Blots relating to Figure 1.
P3shRNA+EV P3shRNA+P3s P3shRNA+P3l P3shRNA+P3sF P3shRNA+P3lF P3shRNA+P3sE P3shRNA+P3lE Annexin V positive ( %) Fig. 2b. Fig. 2d. IP: Caspase 8 Pellino3 shrna knockdown TNFα+ CHX: 5 5hr + Z-VAD-FMK : + + + + + + + + TNFα+ CHX : 0 5 0 5 0 5 0 5hr Caspase-3 Caspase-8 40 TNF + CHX 5hr *** ns *** Caspase 8 18 30 10 0 IP: Caspase 8 Pellino3 shrna knockdown Fig. 2c. + Z-VAD-FMK : + + + + + + + + TNFα+ CHX : 0 5 0 5 0 5 0 5hr Pellino3 Caspase-3 Caspase 8 Supplementary Figure S9: Full Blots relating to Figure 2.
Input Input Rip1 input HA input Fig. 3a. HA IP: Fig. 3b. -pulldown Purified P3s: - + - Purified P3l: - - + Purified Rip1: + + + Rip1 Fig. 3c. -pulldown Purified P3smyc: + Purified P3lmyc: + Purified P3sFmmyc: Purified P3lFmmyc: + + HA-Rip1: + + + + + HA P3smyc: + P3sFHAmmyc: + P3lmyc : + P3lFHAmmyc : + HARip1 : + + + + + P3l P3l P3s Fig. 3d. Input IP: Flag Procaspase-8myc : + + + + Caspase-8DEDmyc : + + + + aspase-8caspasemyc + + Pellino3sflag : + + + + + + Fig. 3e. Input IP: Pellino3smyc: + + Pellino3sFHAmmyc: + + Pellino3lmyc : + + Pellino3lFHAmmyc : + + HAcaspase 8: + + + + + + + + Actin β-actin Procaspase-8 IgG H Caspase-8Caspase Caspase-8DED HA Flag IgG H Pellino3s Fig. 3f. Purified P3smyc: + Purified P3lmyc: + Purified procaspase-8: + + + Caspase 8 -pulldown Fig. 3g. -pulldown Purified P3smyc: + Purified P3lmyc: + Purified Caspase-8Caspase: + + + Caspase 8 30 Pellino3l Pellino3s Supplementary Figure S10: Full Blots relating to Figure 3. Pellino3l Pellino3s
Fig. 3h. Flag Pellino3s-flag IgG H Flag- -myc IgG H Pellino3s-myc Pellino3smyc: + + Flag : + + + + Pellino3sflag: + + + + myc: + + Supplementary Figure S10 (continued): Full Blots relating to Figure 3.
Fig. 4a. Z-VAD-FMK : + + + + TNFα + CHX : 0 2 5 5hr IP : Con Fig. 4b. Z-VAD-FMK : + + + + TNFα + CHX : 0 2 5 5hr IP : C8 C8 C8 Con Pellino3 Pellino3 Fig. 4c. TNFα: 0 5 10 30min IP : TNFR1 TNFR1 TNFR1 TNFR1 28 28 Fig. 4d. IP : Pellino3 Pellino3s EV Pellino3l EV Z-VAD-FMK : + + + + + + + + TNFα + CHX : 0 2 5 5hr 0 2 5 5hr 95 Caspase 8 TRAF2 IgGL TNFR1 Supplementary Figure S11: Full Blots relating to Figure 4.
Fig. 4e. IP : Fig. 4f. IP : P3sFHAm P3s P3lFHAm P3l Z-VAD-FMK : + + + + + + + + TNFα + CHX : 0 2 5 5hr 0 2 5 5hr P3sE3m EV P3lE3m EV Z-VAD-FMK : + + + + + + + + TNFα + CHX : 0 2 5 5hr 0 2 5 5hr 95 95 Caspase 8 Caspase 8 Supplementary Figure S11 (continued): Full Blots relating to Figure 4.
Fig. 5. IP: Z-VAD-FMK : + + + + + + + + + + TNFα+ CHX : 0 4 0 4 0 4 0 4 0 4hr Caspase 8 52 Flag Pellino3 Pellino3Δc Supplementary Figure S12: Full Blots relating to Figure 5.
Fig. 6c. TNF+CHX10hr + + Fig. 6f. Pellino3 -/- MEF TNFα+ CHX 10hr Retro Retro Retro Retro Vector mp3 mp3fm mp3em Caspase 3 Caspase 3 Caspase 3 Caspase 3 β-actin myc Actin GFP 30 Supplementary Figure S13: Full Blots relating to Figure 6.
Supplementary Figure S14: Full Blots relating to Figure 7. Fig. 7b. Fig. 7d. TNF + + Smac + + + + Control shrna Pellino3 shrna TNF + + Smac + + + + Caspase 3 Caspase 3 Pan- ciap Caspase 3 65 Pan-cIAP 65 β-actin β-actin Fig. 7f. Control sirna sirna TNFα+CHX 10hr + + + + + + + + Z-VAD-FMK + + + + Pellino3 +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- Short exposure Long exposure 30 Actin
Fig. 8a. TNFα 0 15 60 1 300 480 0 15 60 1 300 480 min Fig. 8c. P-IKBα TNFα 0 15 60 1 300 480 0 15 60 1 300 480min IKBα Bcl-x 28 p-p38 38 p-jnk 38 Actin P38 JNK 38 p-erk 40 ERK 40. Actin Supplementary Figure S15: Full Blots relating to Figure 8.
Fig. 9c EV TNF + + + + CHX + + IκBα SR TNF + + + + CHX + + β-actin IκBα SR β-actin Supplementary Figure S16: Full Blots relating to Figure 9c.
IP: TNFR1 Supplementary Figure S17: Full Blots relating to Figure 10. IP: Fig. 10a. TNFα: 0 5 10 30 0 5 10 30min Fig. 10b. TNFα: 0 5 10 0 5 10 min 94 TRAF2 Ub TAK1 65 NEMO TNFR1 Fig. 10c. IP: Fig. 10d. IP: TNFα : 0 0 600 0 0 600min Z-VAD-FMK : + + + + TNFα + SMAC : 0 6 0 6hr Pan-cIAP
Fig. 11d. TNFα+ D-gal +/+ -/- Caspase 3 18 Actin Supplementary Figure S18: Full Blots relating to Figure 11d.