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1 DOI: 1.138/n24 Reltive let7i expression Reltive let7i expression CAL27 Twist1 shsr shtwist1 shbmi1 Reltive let7i expression pflagcmv pflagtwist1 Cse 1 Cse 2 BMI1 Eherin Reltive migrtory ility e CMV 1.2 p plet7i pspgtrl pspglet7i let7i reporter Fol hnge of Lu/βgl CAL27 CAL27 mirorna ining sites poly(a) UGAU 3 UUGUCGUGUU GAUGGAGU5 let7i Sponge 5 AACAGCACAA CUACCUCA3 GUCG f mir Reltive let7i expression Fol hnge of Lu/βgl CMV mir1915 CMV Twist11 Twist11 spgtrl mir762 shsr sh sh Twist1 BMI1 let7i mir1915 shsr sh Twist1 spglet7i sh BMI1 Figure S1 Suppression of let7i y Twist1BMI1 in HNSCC ells, n genertion of the let7imniputte stle lones in HNSCC ell lines. () Upper: western lot of Twist1, BMI1, Eherin n in, CAL27, n. tin ws use s loing ontrol. Lower: reltive expression level of let7i in the four ell lines (ompre with ; n=3 for eh ell line). P < 1 y Stuent s ttest. () Upper: phse ontrst imges of HNSCC ell lines in 2D ulture. Sle r=5µm. Lower: Boyen hmer migrtion ssy (ompre with ; n=3 for eh ell line). P < 1 y Stuent s ttest. () Quntittive RTPCR for mir762 n mir1915 in ells trnsfete with Twist1expressing vetor (Twist1) or ontrol vetor (CMV), or in ells trnsfete with shrna ginst Twist1 (shtwist1), BMI1 (shbmi1) or srmle sequene (shsr). n=3 for eh group. P < 1 y Stuent s ttest. () Quntittive RTPCR for let7i expression in two primry HNSCC ultures. Upper: primry HNSCC ulture trnsfete with shrna ginst Twist1 (shtwist1), BMI1 (shbmi1), or srmle ontrol (shsr); lower: primry HNSCC ulture trnsfete with Twist1 or ontrol vetor. n=3 for eh group. P < 1 y Stuent s ttest. (e) Upper: shemti representtion of the let7i sponge vetor; lower: vlition of the let7i sponge vetor y reporter ssy in HEK293T ells. Luiferse tivity/gltosise of ells trnsfete with p n let7i reporter ws pplie s the seline ontrol for the experiments. n=3 for eh group. P < 1 y Stuent s ttest. (f) Upper: quntittive RTPCR nlysis of let7i expression in let7ioverexpresse lones (let7i)(n=3). Trnsfetion of n empty vetor ws use to generte the ontrol lone (). Lower: vlition of the neutrlizing effet of let7i in ells stly expressing the let7i sponge vetor (spglet7i) y reporter ssy (n=3). Trnsfetion of ontrol vetor (spgtrl) ws use s ontrol. P < 1 y Stuent s ttest. The r hrts in pnel ()(f) represent men ± S.E.M. 1
2 let7i e No. of olony let7i spglet7i spgtrl No. of olony let7i Spgtrl 3.E63 2.5E6 2.5 Cell numer(x1 6 ) E62 E6 HRAS E61 LIN28 5.E CMYC E HMGA2 spgtrl Fol hnge of BrDu positive ells spglet7i Om1 let7i1 OL5 let7i2 OL12 4.E54 Cell numer(x1 5 ) 3.E53 E52 E51 CS3 spgtrl 7iS7 spglet7i1 7is22 spglet7i2 E Dy Dy2 Dy3 Dy4 Dy5 Dy Dy2 Dy3 Dy4 Dy5 SSC let7i1 spglet7i1 f let7i IgG 5% 2% CD44 nticd44 56% spgtrl % Fol hnge of BrDu positive ells spgtrl SSC spglet7i1 spgtrl IgG 3% 3% spglet7i CD44 nticd44 16% 45% lone Dose spgtrl spglet7i1 No. of spherois/ 1 ells g let7i No. of spherois/ 1 ells spgtrl spglet7i spglet7i1 1, /6 2/6 1, /6 2/6 1, 2/6 4/6 let7i let7i Eherin h Nonote ish ulture (2D) On top of thik ollgen ulture (2.5D) Emee in ollgen ulture (3D) Eherin i let7i Eherin spglet7i spglet7i spglet7i spgtrl spgtrl spgtrl Eherin Eherin Eherin Figure S2 Suppression of let7i promotes tumorinititing pility without ffeting the expression of EMT mrkers. () Western lot nlysis of HRAS, LIN28, MYC n HMGA2 in OECM1 ells stly trnsfete with let7i expression vetor (let7i) versus ontrol vetor (), or in ells trnsfete with let7i sponge vetor (spglet7i) or ontrol vetor (spgtrl). tin ws use s loing ontrol. () Growth urve nlysis (upper pnels; n=3) n 5romo2 eoxyuriine inorportion ssy (lower pnels; n=3) in n stle ell lines. () Soft gr olony formtion ssy (n=3). P < 1 y Stuent s ttest. () Flow ytometry nlysis of CD44. The perentges of CD44 ells were shown in the right upper qurnt of eh pnel. Isotype IgG ws use s ontrol. (e) Spheroi formtion ssy. Upper: representtive pitures of the spheroi in ifferent ells. Sle r = 1 µm. Lower: quntifition of spherois (n=3). P < 1 y Stuent s ttest. (f) Nue mie xenotrnsplnttion ssy. Left: representtive piture of nue mie reeiving suutneous injetions. Blk rrow inites the formtion of xenotrnsplnte tumor. Cell ose: 1 x 1 4 / mouse. Right: tle showing the result of xenotrnsplnttion stuy. (gi) Western lot nlysis of EMT mrkers (Eherin n ) in let7i vs. n spglet7i vs. spgtrl ultivte on nonote ishes (2D ulture)(g), on top of thik ollgen (2.5D ulture) (h), n emee in ollgen (3D ulture)(i). tin ws use s loing ontrol. The r hrts in pnel (), (), (e) represent men ± S.E.M. 2
3 Nonote ish ulture (2D) On top of thin ollgen ulture CAL27 CAL27 On top of thik ollgen ulture (2.5D) CAL27 On top of thik Mtrigel ulture CAL27 e Emee in ollgen ulture (3D) CAL27 f Nonote ish ulture (2D) g On top of thin ollgen ulture h spgtrl spgtrl On top of thik ollgen ulture (2.5D) spgtrl let7i1 spglet7i1 let7i1 spglet7i1 let7i1 spglet7i1 Figure S3 Phse ontrst imges of HNSCC ells ulture in ifferent onitions. (e) Imges of four HNSCC lines (, CAL27,, n ) ulture in nonote ishes (2D) (), thin ollgenote ishes (), on top of thik ollgen (2.5D)(), on top of thik Mtrigel (), or emee in ollgen (3D)(e). (fh) Imges of let7i vs. n spglet7i vs. spgtrl ulture in nonote ishes (2D) (f), thin ollgenote ishes (g), n on top of thik ollgen (2.5D) (h). Sle r=5µm. 3
4 let7i1 let7i ROCKERKD let7i ROCKERWT 4OHT pmlc2 let7i ROCKERKD phse Ftin/DAPI Ftin/DAPI Ftin/DAPI (single setion) (3D reonstrution) MLC2 let7i ROCKERWT Rtio of perimeter 2 / 4π to re let7i ROCKERKD let7i ROCKERWT µm/min let7i ROCKERKD let7i ROCKERWT Figure S4 let7i reues motility of elongte ells without enhning roune ell movement, n ROCK tivtion hnges neither ellulr shpe nor motility of let7iexpressing ells. () Western lot nlysis of phosphorylte MLC2 (pmlc2) n MLC2 in let7i1 ells, let7i1 ells trnsfete with the 4hyroxytmoxifen (4OHT)inuile vetor whih expresses kinsee ROCKestrogen reeptor fusion protein (ROCKERKD) or wiltype ROCKestrogen reeptor fusion protein (ROCK ERWT), with or without 4OHT inution. tin ws use s loing ontrol. The ells were hrvest from ontop thik ollgen ulture (2.5D) for experiments. () Imges of ells in 2.5D ulture. Representtive setion of phse ontrst mirosopy (sle r=25µm), onfol mirosopy with low power fiel (sle r=25µm), high power fiel (sle r=1µm), n the reonstrutive imges (sle r=1µm) in let7i trnsfete with the vetor expressing kinse e (upper pnels) or wiltype ROCKER fusion protein (lower pnels) fter 4OHT inution for 24 hr. The green olor inites the Ftins stine y n ntiphlloiin ntioy, n lue olor inites the nulei stine y DAPI. () Quntifition of ellulr morphology in let7i expressing ROCKERKD or ROCK ERWT fter 4OHT inution. The morphologi inex ws lulte s perimeter 2 /4πre (n=2 for eh group). () Representtive trjetories n quntifition of motility spee in let7i expressing ROCK ERKD or ROCKERWT fter 4OHT inution in 2.5D ulture (n=1 for eh group). The spee ws lulte s µm/min. The frtion of iniviul ells of let7i ells trnsfete with ROCKERKD n ROCKERWT is 92.8% n 91.6%, respetively. The ox plots in pnel (), () represent smple mximum (upper en of whisker), upper qurtile (top of ox), mein (n in the ox), lower qurtile (ottom of ox), n smple minimum (lower en of whisker). 4
5 Fol hnge of mrna expression let7i1 let7i2 FARP1 ARH GEF15 VAV3 Fol hnge of mrna expression spgtrl spglet7i1 spglet7i2 FARP1 ARH GEF15 VAV3 Collgen emee 3D ulture Collgen emee 3D ulture let7i spglet7i spgtrl 2 GTPRAC1 Totl RAC1 GTPRAC1 Totl RAC1 Figure S5 let7i ownregultes the expression level of mrna n protein. (, ) Quntittive RTPCR nlysis of FARP1, ARHGEF, VAV3, n expression in ells trnsfete with the let7i expression vetor (let7i) or ontrol vetor () (), trnsfete with let7i sponge vetor (spglet7i) or ontrol sponge vetor (spgtrl)(). n=3 for eh group. P < 1 y Stuent s ttest. (, ) Expression of GTPoun RAC1, totl RAC1, n in let7i vs. () n spglet7i vs. spgtrl (). The ells were hrveste from ollgenemee ulture (3D ulture). Totl RAC1 ws pplie s ontrol of tivte RAC1, n tin ws use s loing ontrol. The r hrts in pnel (), () represent men ± S.E.M. 5
6 2 Collgen emee 3D ulture Twist1 Nonote ish ulture (2D) FC FT FTM FTL GTPRAC1 Totl RAC1 Eherin On top of thik ollgen ulture (2.5D) 2.5D Eherin Ftin /DAPI Ftin /DAPI e HRAS 3D Ftin /DAPI Ftin /DAPI f FC IgG 1% nticd44 14% FTM IgG 2% nticd44 31% g lone ose SSC FT 3% 33% SSC FTL 1% 18% 1, 5/6 6/6 6/6 4/6 1, 3/6 5/6 4/6 3/6 CD44 CD44 1, /6 3/6 3/6 /6 Figure S6 Phenotypi impt of Twist1let7i, n HRAS xis on HNSCC ells uner ifferent environments. () Expression of GTPoun RAC1, totl RAC1, Twist1, n in ells trnsfete with pflagcmv (FC), pflagtwist1 (FT), pflagtwist1 n (FTM), n pflagtwist1 n plet7i (FTL). The ells were hrveste from ollgenemee ulture (3D ulture). Totl RAC1 ws pplie s ontrol of tivte RAC1, n tin ws use s loing ontrol. () Upper: phse ontrst imges of FC, FT, FTM n FTL ells in 2D ulture. Sle r=5µm. Lower: western lot of Eherin n in FC, FT, FTM n FTL ells hrveste from 2D ulture. tin ws use s loing ontrol. () Western lot of Eherin n in FC, FT, FTM n FTL ells hrveste from ontop thik ollgen ulture (2.5D). tin ws use s loing ontrol. () Immunofluoresene to show the ellulr morphology n tin orgniztion of FC, FT, FTM, n FTL ells in 2.5D or 3D ulture. The green olor inites the Ftin stine y n ntiphlloiin ntioy, n lue olor inites the nulei stine y DAPI. Sle r = 25µm (first n thir row), 1µm (seon n fourth row). (e) Western lot nlysis of HRAS in FC, FT, FTM, n FTL ells. tin ws use s loing ontrol. (f) Flow ytometry nlysis of CD44. The perentges of CD44 ells were shown in the right upper qurnt of eh pnel. Isotype IgG ws use s ontrol. (g) A tle showing the results of nue mie xenotrnsplnttion ssy. 6
7 EV Snil Snil EV Slug Slug EV Ze1 Ze1 EV Ze2 Ze2 of let7i shsr shsnil Snil of let7i shsr shslug Slug of let7i shsr shze1 Ze1 of let7i shsr shze2 Ze2 of let7i.5 of let7i.5 of let7i.5 of let7i.5 Figure S7 Expression of EMT regultors Snil, Slug, Ze1 or Ze2 oes not signifintly ffet the expression of let7i, n in HNSCC ells. () Upper: western lot nlysis of the expression of the EMT regultor (Snil, Slug, Ze1 or Ze2), n in ells trnsfete with the EMT regultor expression vetor or n empty vetor (EV). The ells were hrveste from ontop thik ollgen ulture (2.5D ulture); lower: quntittive RTPCR nlysis of let7i expression in ells trnsfete with the EMT regultor expression vetor or n empty vetor. n=3 for eh group. P < 1 y Stuent s ttest. () Upper: western lot nlysis of the expression of the EMT regultor (Snil, Slug, Ze1 or Ze2), n in ells reeiving shrna ginst ifferent EMT regultors or srmle sequene. The ells were hrveste from ontop thik ollgen ulture (2.5D ulture); lower: quntittive RTPCR nlysis of let7i expression in ells reeiving shrna ginst ifferent EMT regultors or srmle sequene. n=3 for eh group. P < 1 y Stuent s ttest. The r hrts in pnel (), () represent men ± S.E.M. 7
8 Proportion of overll survivl P=.11 1 Twist1 fol hnge < 2x (n=3) Twist1 fol hnge 2x (n=28) Proportion of isesefree Twist1 fol hnge < 2x (n=3) Twist1 fol hnge 2x (n=28) P= Proportion of overll survivl P=.16 1 fol hnge < 2x (n=24) fol hnge 2x (n=) Proportion of isesefree fol hnge < 2x (n=24) fol hnge 2x (n=) P= Proportion of overll survivl P=.14 1 let7i fol hnge >.5x (n=32) let7i fol hnge.5x (n=) Proportion of isesefree let7i fol hnge >.5x (n=32) let7i fol hnge.5x (n=) P= Proportion of overll survivl P=8 1 2 Twist1negtive (n=64) Twist1positive (n=61) Proportion of isesefree P=6 1 Twist1negtive (n=64) Twist1positive (n=61) e Proportion of overll survivl P=8 1 2 negtive (n=7) positive (n=) Proportion of isesefree P=5 1 2 negtive (n=7) positive (n=) f Proportion of overll survivl P=7 1 2 negtive (n=45) positive (n=8) Proportion of isesefree P=6 1 2 negtive (n=45) positive (n=8) Figure S8 Survivl nlysis of two inepenent sets of HNSCC ptients. () Anlysis of HNSCC ptients group A exmine y quntittive RTPCR (n=58). () Comprison of the overll survivl (; left) n isesefree survivl (; right) of ptients with or without n inrese Twist1 mrna expression (tumor/norml 2 fols). () Comprison of the (left) n (right) of ptients with or without n inrese mrna expression (tumor/norml 2 fols). () Comprison of the (left) n (right) of ptients with or without erese let7i expression (tumor/norml.5 fol). (f) Anlysis of HNSCC ptients group B exmine y immunohistohemistry (n=125). () Comprison of the overll survivl (; left) n isesefree survivl (; right) of ptients with or without positive Twist1 expression. (e) Comprison of the (left) n (right) of ptients with or without positive expression. (f) Comprison of the (left) n (right) of ptients with or without positive expression. The ptients numer (n) of eh group is shown in eh pnel. P vlue ws estimte y logrnk test n is shown in the left lower qurnt of eh pnel. 8
9 shsr shtwist1 IB: Twist1 Figure 1 shsr shbmi1 IB: BMI1 CMV Figure 1 Twist1 Twist1 shsr IB: Twist1 IB: BMI1 Twist1 shbmi1 IB: IB: IB: Figure 3 let7i let7i Spgtrl spglet7i Spgtrl spglet7i IB: GTPRAC1 IB: pmlc2 IB: GTPRAC1 IB: pmlc2 IB: totl RAC1 IB: GTPRho IB: MLC2 IB: IB: totl RAC1 IB: GTPRho IB: MLC2 IB: IB: totl Rho IB: totl Rho 2 let7i IB: IB: IB: VAV3 IB: ARHGEF15 let7i IB: FARP1 IB: Figure 3 2 Spgtrl spglet7i IB: IB: IB: VAV3 IB: ARHGEF15 spglet7i Spgtrl IB: FARP1 IB: Figure S9 Unroppe films showing the key experiments isplye in the min figures. 9
10 Figure 3f Figure 4 let7i1 pdna3 EV IB: IB: 2 let7i1 HAmok IB: HA IB: 1 1 IB: GTP RAC1 IB: Totl RAC1 2 IB: IB: 1 IB: GTPRAC1 1 IB: GTPRAC1 IB: Twist1 IB: 1 IB: Totl RAC1 IB: Totl RAC1 shsr Figure 4 shtwist1 Figure 4 CMV Twist1 Twist1 shsr Twist1 shbmi1 IB: Twist1 IB: IB: 2 IB: 2 IB: IB: IB: Figure S9 ontinue 1
11 Supplementry Movie Legens Movie S1 Timelpse vieo mirosopy of ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. The vieo for spgtrl ws not shown sine the presenttion ws very lose to wiltype ells. Movie S2 Timelpse vieo mirosopy of CAL27 ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Movie S3 Timelpse vieo mirosopy of ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Movie S4 Timelpse vieo mirosopy of ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. The vieo for ws not shown sine the presenttion ws very lose to wiltype ells. Movie S5 Threeimensionl reonstrute immunofluoresent stining imge of the, let7i1, spgtrl, n spglet7i1 ells emee in ollgen. The green olor inites the Ftin stine y n ntiphlloiin ntioy, n lue olor inites the nulei stine y DAPI. Sle r = 1 μm. Movie S6 Timelpse vieo mirosopy of let7i ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S4. The vieo for let7ipdna3 ells ws not shown sine the presenttion ws very lose to let7i ells. Movie S7 Timelpse vieo mirosopy of spglet7i1 ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S1. Movie S8 Timelpse vieo mirosopy of, let7i1, spgtrl, n spglet7i1 emee in ollgen. The urtion of imge pture ws 8 hr for eh lone. Movie S9 Timelpse vieo mirosopy of reonstitute let7i ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S6. Movie S1 Timelpse vieo mirosopy of reonstitute let7i ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S6. Movie S11 Threeimensionl reonstrute immunofluoresent stining imge of CMV, Twist1, Twist1, n Twist1let7i ells emee in ollgen. The green olor inites the Ftin stine y n ntiphlloiin ntioy, n lue olor inites the nulei stine y DAPI. Sle r = 1 μm. Movie S12 Timelpse vieo mirosopy of CMV ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Movie S13 Timelpse vieo mirosopy of Twist1 ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. The vieos for Twist1 ells, n Twists1 ells trete with vehile ontrol were not shown sine the presenttion ws very lose to Twist1 ells. Movie S14 Timelpse vieo mirosopy of Twist1let7i ells plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S12, S13. Movie S15 Timelpse vieo mirosopy of CMV, Twist1, Twist1, n Twist1let7i ells emee in ollgen. The urtion of imge pture ws 8 hr for eh lone. Movie S16 Timelpse vieo mirosopy of Twist1 ells trete with the RAC1 inhiitor NSC μm for 12 hrs, then plte on top of thik ollgen. The urtion of imge pture ws 24 hr. Compre with Movie S
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