mch log height Counts GFP log height mch log height Counts GFP log height mch log height Counts high flux % Hist: 8.34 EBSS shctrl Counts

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Gerald Cox
5 years ago
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1 DOI:.8/n2886 mchrry-gfp-lc3 Autophgy Rportr: Autophgosom Autolysosom mchrry GFP LC3 mchrry GFP LC3 X ph >6. ph <5. mchrry-gfp-lc3 Clls mch log hight % Hist: % Hist: Lsr Exittion Flow Dttor mch log hight % Hist:.28 % Hist: Cll Sortr Low flux lls High flux lls Trhlos mch log hight % Hist: 3.49 % Hist: shctrl % Hist: 8.79 % Hist: 8. shctrl % Hist:.67 % Hist: shatg % Hist: 2 3. % Hist: 9.42 shatg % Hist: 9.52 % Hist: Liv Cll Fluorsn.6.25 Sponin-Insolul Fluorsn f GFP GFP mchrry.2.4 mchrry Lysotrkr.5 n Fluorsn (Fol ovr ) High Low n Fluorsn (Fol ovr ) High Low GFP utophgosoms (sponin) GFP-Log_Hight mchrry utophgosoms n utolysosoms (sponin) mchrry-log_hight Supplmntry Figur Mtho to sort lls with high n low utophgi flux y flow ytomtry urtly msurs sl n inu utophgi flux., Clls onstitutivly xprssing mchrry-gfp-lc3 r sort s on th rltiv rtio of mchrry/egfp fluorsn whih hngs in rspons to th ph grint s utophgosoms fus with lysosoms to form utolysosoms (utophgi flux)., BJAB mchrry-gfp-lc3 lls wr trt with (%) or trhlos (75 mm) for 4 hours follow y flow ytomtry for utophgi flux. Dt r rprsnttiv of t lst 3 inpnnt xprimnts., HL mchrry-gfp-lc3 lls xprssing ontrol or Atg5 shrna wr trt with for 4 hours follow y flow ytomtry for utophgi flux. Dt r rprsnttiv of t lst 3 inpnnt xprimnts., BJAB mchrry- GFP-LC3 lls wr sort y flow ytomtry for high or low utophgi flux, thn trt with Lysotrkr Blu for 3 min. t 37 C, follow y flow ytomtry (min GFP, mchrry or Lysotrkr Blu fluorsn normliz to fol ovr unsort ontrol, mn ± s..m., n=3 wlls)., BJAB mchrry- GFP-LC3 lls wr sort s in () follow y ytoplsmi xtrtion with. % sponin to limint non-lipit LC3 n flow ytomtry to quntitt utophgosom/utolysosom numr (normliz fol ovr unsort ontrol min GFP or mchrry fluorsn, mn ± s..m., n=3 wlls). f, Flow ytomtry histogrms of rprsnttiv t from () Mmilln Pulishrs Limit. All rights rsrv.

2 Mrg GFP ololiztion mchrry ololiztion Lysotrkr μm Cololiztion Intnsity R-Grn High R-Blu Low % Hist: % Hist: 2.33 SSC-Ar 28 sttr (FL4-Hight/FL-Hight) f FSC-Hight Su-G G S Su-G G S G2/M 2 8 G2/M (SSC-Ar/FSC-Hight) Dpi-Log_Hight Supplmntry Figur 2 Confol mirosopy rvls iffrns in th numr of utophgosoms n utolysosoms in flux sort lls; High n low flux sort lls xhiit similr siz, ll yl n poptoti profil., HL mchrry-gfp-lc3 lls wr sort for utophgi flux, trt with Lysotrkr Blu for 3 min. t 37 C, hr to slis y ytospin ntrifugtion, fix with formlhy n visuliz y spinning is onfol mirosopy; ths r th sm fils pit in Fig.. Cololiztion imgs wr prou using ImgJ (s mthos)., Quntifition of th ololiztion imgs in (). Dt r rprsnttiv of 2 sprt fils n hv n rpt 5 tims., BJAB mchrry-gfp-lc3 lls wr sort for utophgi flux y flow ytomtry.,, Forwr n si sttr of sort lls in (). f, Cll yl profils of sort lls in () stin with Hohst Mmilln Pulishrs Limit. All rights rsrv.

.4.2..8.6.4.2. Fs Lign.4.2..8.6.4.2. TRAIL DAPI log hight 4 3 2 5 375 25 25 -.2 D % Hist: 2.96 Fs Lign.5.5.5 5 5 Untrt Fs Lign (ng/ml) Erly Apoptoti % Hist: 6.82 Non-poptoti % Hist: 88.

3 Fs Lign TRAIL DAPI log hight D % Hist: 2.96 Fs Lign Untrt Fs Lign (ng/ml) Erly Apoptoti % Hist: 6.82 Non-poptoti % Hist: DAPI log hight No Lign 4h 4h 4h Csps-3/7-Log_Hight D % Hist: 5.67 f Fs lign 4 hr Nulr CllEvnt Csps 3/7 (Fol ovr ) Erly Apoptoti % Hist: Non-poptoti % Hist: Csps-3/7 Fluorsn n (Aritrry Units) ng/ml 7.5 ng/ml 2.5 ng/ml 2.5 ng/ml.75 ng/ml.75 ng/ml TRAIL (ng/ml) 2 4 Hours ftr Fs Lign Aition Hours * Supplmntry Figur 3 Autophgy moultion of Fs lign-inu poptosis n ll killing.,, BJAB mchrry-gfp-lc3 lls wr sort for utophgi flux y flow ytomtry follow y trtmnt with Fs lign () or TRAIL () for 24 hours n MTS ssy for viility (fol ovr no lign ontrol, mn ± s..m., n=3 wlls). Ths r th full t sts from th sm xprimnt pit in Figur 2. -, BJAB mchrry-gfp-lc3 lls wr sort s in () follow y trtmnt with Fs lign for th init tims in th prsn of fluorogni Csps-3/7 sustrt CllEvnt Grn (5 μm) n nlyz y flow ytomtry., Dot plot of Csps-3/7 tivity vs. DAPI fluorsn in unsort lls., Quntittion of Csps-3/7 tivity in sort lls t th init tim points (min CllEvnt Grn fluorsn, mn ± s..m., n=3 wlls, *p=.9)., Rprsnttiv flow ytomtry histogrms of Csps-3/7 tivity in (, ). f, BJAB mchrry-gfp-lc3 lls wr sort for utophgi flux s ov, follow y trtmnt with Fs lign t th init onntrtions in th prsn of fluorogni CllEvnt Csps-3/7 sustrt (5 μm) n nlyz on n InuCyt ZOOM for th init tims (mn CllEvnt Grn fluorsn pr fil, mn, n=2: 4 fils for h of 3 wlls) Mmilln Pulishrs Limit. All rights rsrv.

4 .4 +zvad shrna: Atg2 Atg shctrl shctrl-cq shbi shbi-cq Cll Viility (% of ) 8 Chloroquin ** DCMI * Doxoruiin Etoposi TRAIL Fs Lign Atg4DN R7DN NS NS - CQ Atg5 Atg5 - CQ f Trhlos g Asorn (49 nm) Trhlos Fs Lign (ng/ml) Fs Lign (ng/ml) Supplmntry Figur 4 Efft of utophgy inhiition or utophgy inution on Fs lign-inu th., BJAB lls xprssing ontrol or Atg2 shrna wr trt with Fs lign (5 ng/ml) for 24 hours in th prsn or sn of zvad-fmk ( μm). Cll viility ws thn quntitt y MTS sorn (fol ovr untrt ontrol, mn ± s..m., n=3 wlls)., BJAB lls xprssing ontrol or Bi shrna wr trt with utophgy inhiitor hloroquin (25 μm) for 2 hours follow y Fs lign (4 ng/ml) for 24 hours. Cll viility ws thn quntitt y MTS (fol ovr untrt ontrol, mn ± s..m., n=3 wlls)., BJAB lls wr trt with utophgy inhiitors hloroquin (2 μm) or smthyllomiprmin (DCMI, μm) for 2 hours follow y trtmnt with th init ytotoxi gnts for 24 hours. Clls wr thn ssy for viility y MTS (% of ontrol (no ytotoxi rug), mn ± s..m., n=3 wlls, *p=.24, **p=.)., BJAB lls xprssing vtor ontrol or th init utophgy ominnt-ngtiv onstruts wr trt with Fs lign (4 ng/ml) for 24 hours; ll viility ws thn quntitt y MTS sorn (fol ovr untrt ontrol, mn ± s..m., n=3 wlls). Dt r rprsnttiv of 2 inpnnt xprimnts., BJAB lls xprssing ontrol or Atg5 shrna wr trt with hloroquin (2 μm) for 2 hours follow y Fs lign (4 ng/ml) for 24 hours. Cll viility ws thn quntitt y MTS (fol ovr no lign ontrol, mn ± s..m., n=3 wlls). Dt r rprsnttiv of 3 inpnnt xprimnts. f, g, BJAB lls wr trt with th init utophgy inurs ovrnight (, %; trhlos, 75 mm) follow y trtmnt with Fs lign t th init onntrtions for 24 hours. Cll viility ws thn quntitt y MTS sorn. f, MTS t normliz (fol ovr untrt (no Fs lign) ontrol, mn ± s..m., n=3 wlls). g, Rw MTS vlus (sorn t 49 nm, mn ± s..m., n=3 wlls) Mmilln Pulishrs Limit. All rights rsrv.

5 Fs rptor t ll surf shrna: Fp- BJAB Atg5 Atg7 Vps Jurkt Atg5 Atg7 Vps Fp- IB No Lign Fp- IP FsL No Lign FsL APC-αCD APC-Log_Hight log hight Asorn (49 nm) sh shctrl-cq Fp- 88 Fp- 88-CQ Fp- 89 Fp- 89-CQ Asorn (49 nm) sh shctrl CQ Fp- 88 Fp 88-q Fp- 89 Fp- 89-q Fs Lign (ng/ml) TRAIL (ng/ml) Supplmntry Figur 5 Autophgy ontrols ll surf xprssion of Fs rptor vi Fp-., BJAB mchrry-gfp-lc3 lls wr sort for utophgi flux y flow ytomtry, n thn stin with APC-onjugt nti-fs ntioy t 4 C for 3 minuts. Clls wr thn wsh n nlyz y flow ytomtry. Dt r rprsnttiv of 3 rplits from 3 inpnnt xprimnts., Aitionl xposurs of immunolots from xprimnt in Fig. 4f. -, BJAB lls xprssing ontrol or Fp- shrnas wr trt with 2 µm hloroquin for 2 hours follow y trtmnt with Fs lign () or TRAIL () t th init onntrtions for 24 hours. Cll viility ws quntitt y MTS (sorn t 49 nm, mn ± s..m., n=3 wlls). Ths r th full os rspons urvs (without normliztion) from th t in Figur 5., Aitionl xposurs from immunopripittion xprimnt pit in Fig Mmilln Pulishrs Limit. All rights rsrv.

6 Fig. Fig. Fig. Fig. 2 Fig. 2 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4f Fig. 4f Fig. 4f Fig. 4f Fig. 4f Fig. 4f Fig. 4f Fig. 4f Fig. 5 Fig. 5 Fig. 5 Fig. 5 Fig. 5 Fig. 6 Fig. 5 Fig. 5 Fig. 5 Fig. 6 Fig. 6 Fig. 6 Fig. 6 Fig. 6 Fig. 6 Fig. 6 Fig. 6 Supplmntry Figur 6 Full immunolot imgs. R oxs init th ropp portion of h wstrn lot prsnt in th orrsponing min figurs Mmilln Pulishrs Limit. All rights rsrv.

Coatomer LPAAT-γ Merge

Coatomer LPAAT-γ Merge DI: 1.138/n2273 Cotomr LPAAT-γ Mrg Tuuls (%) 1 5 < 5 nm 5~1nm 1~2nm 2~5nm >5nm 5~1nm < 5 nm 1~2nm 2~5nm >5nm 2 min 5 min 1 min 3 min < 5 nm 5~1nm 1~2nm 2~5nm >5nm < 5 nm 5~1nm 1~2nm 2~5nm >5nm Figur S1