A ) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1 . Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) were determined with an automatic flux analyzer ( Seahorse / Agilent ) . After monitoring basal respiration , oligomycin ( 1 µM ) , and rotenone plus antimycin A ( Rot ; 0 . 5 µM ; AA , 0 . 5 µM ) were added sequentially to determine glycolytic and mitochondrial ATP production rates . ~ 12 technical replicates per measurement , N = 4 independent assays .

O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.


A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP - positive cells was determined 16 h later by flow cytometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.


B LDH release from HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . At 24 h post transfection , cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined . Graphs show mean and s . d . of quadruplicate wells .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.


C - D . PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD in the presence of osmoprotectants . At 24 h post transfection , PEGs of the indicated molecular weights were added to a final concentration of 30 mM , cells were treated with 250 ng / ml doxycycline for 8 h and the level of PI staining ( C ) or LDH release ( D ) was determined .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.


E - F . PI staining of and LDH release from LPS - primed primary BMDMs infected with log - phase S . typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight ( numbers on the X - axis , 30 mM final concentration ) . * p < 0 . 05 as determined by student ' s t test . Graphs show mean and s . d . of quadruplicate wells . Data are representative of at least three independent expeirments .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.


A . Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD , cleaved GSDMD and α - tubulin in the cell lysates of immortalized LPS - primed WT macrophages left uninfected ( NS ) or infected for 10 - 40 minutes with log phase S . typhimurium ( MOI = 50 ) .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).


C . Fractionation and immunoblot analysis for GSDMD , Na + K + ATPase , VDAC ( Voltage - dependent anion channel ) , HDAC1 ( Histone deacetylase 1 ) and GAPDH ( Glycerinaldehyd - 3 - phosphat - Dehydrogenase ) of WT macrophages infected for 10 and 20 minutes with log phase S . typhimurium ( MOI = 50 ) . Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane .

O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane.


D . Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S . typhimurium ( MOI = 50 ) . Extraction was carried in variable conditions as described in the Material and Methods section .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.


A . Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . The protein was cleaved in a time - dependent manner into two bands of 31 kDa ( GSDMDNterm ) and 22 kDa ( GSDMDCterm ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O

A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm).


B . Cross - linking experiment of full - length and cleaved GSDMD . GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . After enzymatic cleavage , GSDMDNterm is highly cross - linked by DSS , resulting in the gel - impenetrating species highlighted by the arrow . GSDMDCterm is not cross - linked .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked.


C . GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase - 1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E . coli . After 2 h , the lipid fraction ( L ) was separated from the supernatant ( S ) by ultracentrifugation at 4C for 1 h at 120 , 000g .

O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.


A - I . Dye release time courses from liposomes as a percentage of maximal release . A . Five different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli polar lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 130 , 100 , 65 ( colored dark to light orange ) . The time point of 20 min is highlighted by a vertical dashed line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . B . Five different reactions , where 130 nM of GSDMD and 400μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli extract polar lipid were incubated with caspase - 1 concentration of ( nM ) : 15 , 8 , 5 , 2 . 5 , 1 . 2 ( colored dark to light blue ) . The time point of 20 min is highlighted by a vertical dashed line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . C . Three different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 100 ( colored dark to light green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green).


A - I . Dye release time courses from liposomes as a percentage of maximal release . D . Dye release at 20 minutes reaction as a function of GSDMD ( dark orange ) and caspase - 1 ( dark blue ) concentrations . Errors bars for three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . E . Two different sets of reactions , where wild type GSDMD ( dark to light orange ) and the mutant GSDMDI104N ( dark to light blue ) were independently incubated at the concentrations of 260 , 130 and 65 nM with 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes . The time point of 60 minutes is highlighted by a vertical dashed line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . F . Dye release at 60 minutes of reaction as a function of GSDMD wild type ( dark orange ) and GSDMDI104N ( dark blue ) concentration . Error bars for three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . G - I . Dye release from 400 μM liposomes loaded with the 6 - carboxifluorescein - derivates FD - 20 , FD - 40 and FD - 150 , with variable Stokes diameters , as indicated . 130 nM of GSDMD and 5 nM caspase - 1 were incubated with the liposomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.


A - C . Cryo - EM micrographs of GSDMDNterm pores in E . coli extract polar lipids liposomes . The micrographs were acquired at protein / lipid molar ratios of 1 / 1000 , 1 / 500 and 1 / 100 , respectively . Black arrows indicate ring - shaped structures corresponding to oligomeric GSDMDNterm pore forms . Scale bars = 80 nm .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

A-C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm.


D . Proteoliposome with protein / lipid molar ratio of 1 / 100 at higher magnification . Scale bar = 80 nm .

O O O O O O O O O O O O O O O O O O O O O O O

D. Proteoliposome with protein/lipid molar ratio of 1/100 at higher magnification. Scale bar = 80 nm.


A . AFM topograph of GSDMDNterm bound to lipid membranes of E . coli extract polar lipids . Overview topograph showing arc - , slit - and ring - like GSDMD oligomers .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers.


B - E . High - resolution AFM topographs and height profiles of GSDMDNterm oligomers ( red curves ) . Height profiles were measured along the red lines in the topographs . Dashed zero lines ( 0 nm ) indicate the surface of the lipid membrane ; heights of < − 4 nm indicate that the oligomers formed transmembrane pores . Topographs were taken in buffer solution ( 150 NaCl , 20 mM Hepes , pH 7 . 8 ) at room temperature . The experiment was reproduced at least three times using independent liposome and GSDMD preparations .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

B-E. High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves). Height profiles were measured along the red lines in the topographs. Dashed zero lines (0 nm) indicate the surface of the lipid membrane; heights of <−4 nm indicate that the oligomers formed transmembrane pores. Topographs were taken in buffer solution (150 NaCl, 20 mM Hepes, pH 7.8) at room temperature. The experiment was reproduced at least three times using independent liposome and GSDMD preparations.


F - H . Analysis of GSDMDNterm oligomers bound to lipid membranes of E . coli extract polar lipids . F . GSDMDNterm oligomers assembled into arcs , slits and rings leading to transmembrane ( TM ) pores or not transmembrane aggregate ( n = 218 ) . G . Height of GSDMDNterm oligomers protruding from the lipid membrane . Bars represent mean and error bars represent s . e . m . ( n = 218 ) . H . Distribution of the diameters of rings formed by GSDMDNterm oligomers . The average distribution was 21 . 2 ± 5 . 6 nm ( n = 164 ; average ± s . d . ) and the bin size was 3 nm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.


( E ) Expression levels of LSD1 in MGC - 803 , MKN - 45 , HGC - 27 , BGC - 823 , and NCI - N87 cell lines and their corresponding sEVs . The samples with equal amounts of proteins were loaded ( n = 3 biological replicates ; mean ± SEM ; compared with the NCI - N87 ; * P = 0 . 0432 ( cell / MGC - 803 ) , * P = 0 . 0300 ( cell / HGC - 27 ) , * * P = 0 . 0306 ( cell / BGC - 823 ) , * * P = 0 . 0018 ( sEVs / MGC - 803 ) , * * P = 0 . 0029 ( sEVs / HGC - 27 ) , and * * * P = 0 . 0003 ( sEVs / BGC - 823 ) ; two - tailed unpaired Student ' s t - test ; GAPDH was used as a loading control for cell lysis ; CD9 was used as a loading control for sEV lysis ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *P = 0.0432 (cell/MGC-803), *P = 0.0300 (cell/HGC-27), **P = 0.0306 (cell/BGC-823), **P = 0.0018 (sEVs/MGC-803), **P = 0.0029 (sEVs/HGC-27), and ***P = 0.0003 (sEVs/BGC-823); two-tailed unpaired Student's t-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis).


( J ) Expression levels of LSD1 , CD63 , and CD9 in sEVs with indicated treatment .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

(J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.


( A ) Confocal microscopy image analysis of sEV fusion to MGC - 803 cells . The MGC - 803 ( left side ) and MKN - 45 ( right side ) cells were treated with sEVs derived from MGC - 803 cells ( Con sEVs ) or LSD1 knockout ( KO ) MGC - 803 cells ( KO sEVs ) and stained with PKH26 for 12 h . Additionally , the cell membrane was stained with Dio , while the nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.


( B ) Immunofluorescence confocal microscopy analysis of LSD1 ( green ) in LSD1 KO MGC - 803 cells ( left panel ) and LSD1 KO MKN - 45 cells ( right panel ) incubated with 20 μg / mL Con sEVs and KO sEVs for 12 h . The cell membrane was stained with PKH26 , while the nuclei were stained with DAPI . Scale bar = 100 µm .

O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.


( F ) Expression levels of LSD1 , Nanog , OCT4 , SOX2 , and CD44 in LSD1 KO MGC - 803 cells .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O

(F) Expression levels of LSD1, Nanog, OCT4, SOX2, and CD44 in LSD1 KO MGC-803 cells.


( N ) Immunoprecipitation of Kme1 / 2 on SOX2 ( WT indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - WT ; Mut indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - Mut ; WT , wild type ; Mut , mutant ) .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).


( C ) Expression levels of LSD1 , OCT4 , and SOX2 in MGC - 803 and MKN - 45 cells incubated with 20 μg / mL sEVs from MGC - 803 or LSD1 KO MGC - 803 cells for 48 h . ( D ) Quantification of the results of ( C ) ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0385 ( LSD1 ) , * P = 0 . 0160 ( OCT4 ) , and * * * P = 0 . 0006 ( SOX2 ) for MGC - 803 ; * P = 0 . 0299 ( LSD1 ) , * P = 0 . 0147 ( OCT4 ) , and * P = 0 . 0258 ( SOX2 ) for MKN - 45 ; two - tailed unpaired Student ' s t - test ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h. (D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *P = 0.0385 (LSD1), *P = 0.0160 (OCT4), and ***P = 0.0006 (SOX2) for MGC-803; *P = 0.0299 (LSD1), *P = 0.0147 (OCT4), and *P = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's t-test).


( F ) Sphere formation assay results of MGC - 803 ( upper panel ) and MKN - 45 ( bottom panel ) cells incubated with 20 μg / mL sEVs from HEK293T cells ( left panel ) , WT - LSD1 sEVs ( middle panel ) , and LSD1 K661A sEVs ( right panel ) for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0021 ( MGC - 803 ) and * * P = 0 . 0020 ( MKN - 45 ) ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0021 (MGC-803) and **P = 0.0020 (MKN-45); two-tailed unpaired Student's t-test).


( F ) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay . Scale bar = 50 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0024 , * * P = 0 . 0088 , and * P = 0 . 0342 ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).


( I and J ) Expression levels of LSD1 in sEVs isolated from the plasma . CD9 and CD63 were used as markers of sEVs . CD9 was used as a loading control for sEV lysis ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0256 ( patient 1 ) , * * P = 0 . 0068 ( patient 2 ) , * * P = 0 . 0013 ( patient 3 ) , * * P = 0 . 0031 ( patient 4 ) , * * * P = 0 . 0009 ( patient 5 ) , * * P = 0 . 0012 ( patient 6 ) , * * * P = 0 . 0007 ( patient 7 ) , * * * P = 0 . 0010 ( patient 8 ) , * * * P = 0 . 0001 ( patient 9 ) , and * P = 0 . 0461 ( patient 10 ) ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *P = 0.0256 (patient 1), **P = 0.0068 (patient 2), **P = 0.0013 (patient 3), **P = 0.0031 (patient 4), ***P = 0.0009 (patient 5), **P = 0.0012 (patient 6), ***P = 0.0007 (patient 7), ***P = 0.0010 (patient 8), ***P = 0.0001 (patient 9), and *P = 0.0461 (patient 10); two-tailed unpaired Student's t-test).


( K ) Sphere formation assay results of MGC - 803 cells treated with sEVs as indicated for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0019 and * * P = 0 . 0050 ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).


Proliferation assay results of MGC - 803 ( A ) , MKN - 45 ( B ) , NCI - N87 ( C ) cells treated with oxaliplatin along with indicated treatments ( n = 3 biological replicates , mean ± standard error of mean ( SEM ) ; * P = 0 . 0204 ( MGC - 803 ) , * P = 0 . 0498 ( MKN - 45 ) , * P = 0 . 0473 ( NCI - N87 ) , and * P = 0 . 0019 ( LSD1 KO MGC - 803 ) ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).


Representative images of tumors ( E ) and the tumor weight ( F ) of mice treated with oxaliplatin in the presence or absence of sEVs ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0488 ; two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).


Loss of LRRC8D causes resistance to carboplatin and cisplatin , but not to oxaliplatin . Survival of parental , vector ‐ transduced , or LRRC8D ‐ deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin , carboplatin , and oxaliplatin . The corresponding IC50 values and 95 % confidence interval ( CI ) are given in Appendix Table S2 . Data are presented as mean ± SEM .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.


A , BVRAC currents ( ICl , vol ) of the HAP1 ( A ) and KBM7 ( B ) haploid cell lines . Left panels , example current traces of ICl , vol fully activated by hypotonic cell swelling measured with the voltage ‐ clamp protocol shown in ( A ) . Dashed lines indicate zero current . Right panels , averaged current / voltage relationships of maximally activated ICl , vol . Consistent with VRAC currents , they needed hypotonic swelling for activation , displayed an I − > Cl − permeability sequence , and were blocked by DCPIB ( Appendix Fig S2A - H ) . The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E ( Fig EV1 ) which accelerates VRAC inactivation ( Voss et al , 2014 ) . At potentials > + 100 mV , also KBM7 currents inactivated ( Appendix Fig S2I ) . Data are presented as mean ± SEM ; n = 5 - 10 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, BVRAC currents (ICl,vol) of the HAP1 (A) and KBM7 (B) haploid cell lines. Left panels, example current traces of ICl,vol fully activated by hypotonic cell swelling measured with the voltage‐clamp protocol shown in (A). Dashed lines indicate zero current. Right panels, averaged current/voltage relationships of maximally activated ICl,vol. Consistent with VRAC currents, they needed hypotonic swelling for activation, displayed an I− > Cl− permeability sequence, and were blocked by DCPIB (Appendix Fig S2A-H). The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E (Fig EV1) which accelerates VRAC inactivation (Voss et al, 2014). At potentials > +100 mV, also KBM7 currents inactivated (Appendix Fig S2I). Data are presented as mean ± SEM; n = 5-10.C


CDependence of regulatory volume decrease ( RVD ) of HEK cells on LRRC8 genes . Cells were exposed to hypotonic medium starting at t = 0 , and intracellular calcein fluorescence was followed over ˜ 1 h as semiquantitative measure of cell volume . Data are presented as mean values ± SEM from sixteen wells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CDependence of regulatory volume decrease (RVD) of HEK cells on LRRC8 genes. Cells were exposed to hypotonic medium starting at t = 0, and intracellular calcein fluorescence was followed over ˜1 h as semiquantitative measure of cell volume. Data are presented as mean values ± SEM from sixteen wells.


LRRC8 subunit ‐ and osmolarity ‐ dependent caspase induction in HCT116 cellsA , BCisplatin ‐ induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions ( A ) , or after 1 h exposure to 200 μM cisplatin under iso ‐ and hypotonic conditions ( B ) , was followed over time in WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Results from HAP1 − / − and LRRC8D − / − were obtained with two different clonal cell lines each and averaged . CCaspase activation after 1 ‐ h exposure to 4 μM staurosporine under iso ‐ or hypotonic conditions of WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Data information : Data are presented as mean ± SEM , n = 3 - 6 . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . Similar results were obtained in three independent experiments . Fold change in ( A ) refers to t = 0 . Control experiments indicated that hypotonicity per se had no effect ( Appendix Fig S4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LRRC8 subunit‐ and osmolarity‐dependent caspase induction in HCT116 cellsA, BCisplatin‐induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions (A), or after 1 h exposure to 200 μM cisplatin under iso‐ and hypotonic conditions (B), was followed over time in WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells. Results from HAP1−/− and LRRC8D−/− were obtained with two different clonal cell lines each and averaged.CCaspase activation after 1‐h exposure to 4 μM staurosporine under iso‐ or hypotonic conditions of WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells.Data information: Data are presented as mean ± SEM, n = 3-6. *P < 0.05; **P < 0.01; and ***P < 0.001. Similar results were obtained in three independent experiments. Fold change in (A) refers to t = 0. Control experiments indicated that hypotonicity per se had no effect (Appendix Fig S4).


Activation of LRRC8 channels by pro ‐ apoptotic stimuliA - CCisplatin ‐ induced iodide influx into WT ( black ■ ) , but not HAP1 − / − ( green ▼ ) HEK cells indicates VRAC halide current activation during apoptosis . Cells expressing an iodide ‐ sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0 . 5 h ( A ) , 4 . 5 h ( B ) , or 8 . 5 h ( C ) before adding extracellular I − ( 50 mM final ) . The difference in slopes of YFPfluorescence quenching between control and cisplatin ‐ treated cells semiquantitatively reflects VRAC current activation . Note that increased YFP quenching with cisplatin preincubation is not due to large non ‐ specific leaks resulting from cell morbidity . Such leaks should lead to a fast component of YFP quenching in WT , but not HAP1 − / − cells after the pipetting artifact that immediately follows addition of iodide ( indicated by arrows ) . DSwelling ‐ induced iodide influx into WT ( black ■ ) and HAP1 − / − ( green ▼ ) HEK cells for comparison . Iodide ( 50 mM final ) was added in isotonic or hypotonic ( 230 mOsm final ) solution at the time indicated by arrow . E , FTime course of VRAC activation by 200 μM cisplatin ( E ) or 4 μM staurosporine ( F ) determined as in ( A - C ) . Averaged maximal slopes of YFP quenching from eight wells ( E ) or 16 wells ( F ) each were evaluated to estimate iodide influx rates . WT ( black ■ ) and HAP1 − / − ( green ▼ ) . Data are presented as mean ± SEM .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Activation of LRRC8 channels by pro‐apoptotic stimuliA-CCisplatin‐induced iodide influx into WT (black ■), but not HAP1−/− (green ▼) HEK cells indicates VRAC halide current activation during apoptosis. Cells expressing an iodide‐sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0.5 h (A), 4.5 h (B), or 8.5 h (C) before adding extracellular I− (50 mM final). The difference in slopes of YFPfluorescence quenching between control and cisplatin‐treated cells semiquantitatively reflects VRAC current activation. Note that increased YFP quenching with cisplatin preincubation is not due to large non‐specific leaks resulting from cell morbidity. Such leaks should lead to a fast component of YFP quenching in WT, but not HAP1−/− cells after the pipetting artifact that immediately follows addition of iodide (indicated by arrows).DSwelling‐induced iodide influx into WT (black ■) and HAP1−/− (green ▼) HEK cells for comparison. Iodide (50 mM final) was added in isotonic or hypotonic (230 mOsm final) solution at the time indicated by arrow.E, FTime course of VRAC activation by 200 μM cisplatin (E) or 4 μM staurosporine (F) determined as in (A-C). Averaged maximal slopes of YFP quenching from eight wells (E) or 16 wells (F) each were evaluated to estimate iodide influx rates. WT (black ■) and HAP1−/− (green ▼). Data are presented as mean ± SEM.


Carboplatin uptake into control KBM7 cells and two LRRC8D ‐ deficient clones ( n = 6 ) .

B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).


Hypotonicity ‐ stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone ( CBX ) , a non ‐ specific blocker of VRAC ( n = 3 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).


Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long ‐ term isotonic ( C ) or 200 μM cisplatin in short ‐ term hypo ‐ and isotonic ( D ) conditions as function of time ( n = 3 ) . Similar results were obtained in HCT116 cells ( Fig EV4 ) .

B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).


Cisplatin uptake ( 200 μM ) into HEK cells of indicated genotypes . LRRC8 ( B , C , E ) − / − and LRRC8 ( B , D , E ) − / − cells express only LRRC8A and LRRC8D , and LRRC8A and LRRC8C , respectively ( n = 3 for WT , 6 for LRRC8D − / − , 9 for LRRC8A − / − in E ; n = 3 in F ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F).


Mean current densities of maximally activated ICl , vol at − 80 mV . The number of cells is indicated for each column .

O O O O O O O O O O O O O O O O O O O O O O O O

Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column.


Ratio of LRRC8 ‐ dependent swelling ‐ activated cisplatin uptake ( 60 min ) to mean ICl , vol ( as in G ) as function of genotype . Data information : Data are presented as mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 ( for ( C , E ) compared to HAP1 − / − cells ) . Dotted lines in ( D ) and ( F ) highlight that there is no significant difference in isotonic 60 ‐ min uptake between the genotypes .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.


Cisplatin - DMSO inhibition of ICl , vol depends on the LRRC8D subunitUpper panel , example current traces ( as in Fig 3A ) of fully activated ICl , vol in HEK cells exposed to hypotonic solution containing vehicle ( 0 . 3 % DMSO ) or 200 μM cisplatin in 0 . 3 % DMSO . Dashed lines indicate zero current . Lower panel , ICl , vol current densities ( at − 100 mV and 100 mV ) of WT HEK cells treated with different cisplatin concentrations . No effect of 200 μM cisplatin / DMSO on ICl , vol in LRRC8D − / − HEK cells . Data information : Data are presented as mean ± SEM ; the number of experiments is given for each bar ; * P < 0 . 05 ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cisplatin-DMSO inhibition of ICl,vol depends on the LRRC8D subunitUpper panel, example current traces (as in Fig 3A) of fully activated ICl,vol in HEK cells exposed to hypotonic solution containing vehicle (0.3% DMSO) or 200 μM cisplatin in 0.3% DMSO. Dashed lines indicate zero current. Lower panel, ICl,vol current densities (at −100 mV and 100 mV) of WT HEK cells treated with different cisplatin concentrations.No effect of 200 μM cisplatin/DMSO on ICl,vol in LRRC8D−/− HEK cells.Data information: Data are presented as mean ± SEM; the number of experiments is given for each bar; *P < 0.05; **P < 0.01.


Swelling ‐ induced efflux of 3 [ H ] ‐ taurine from WT and LRRC8D − / − HEK cells ( A , B ) and partial rescue by transient transfection of LRRC8D ( B ) . Rescue is incomplete due to low transfection / expression efficiency of LRRC8D ( Voss et al , 2014 ) .

O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D −/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).


C - ESwelling ‐ induced efflux of 3 [ H ] ‐ taurine from LRRC8 ( B , C , E ) − / − ( C ) , LRRC8 ( B , D , E ) − / − ( D ) , or LRRC8 ( B , C , D ) − / − ( E ) HEK cells compared to WT cells . FCisplatin ‐ induced taurine efflux ( over 30 min ) from WT and HAP1 − / − HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h , or without cisplatin ( 0 h ) . Carbenoxolone ( CBX ; 100 μM ) blocks taurine efflux from WT cells treated for 12 h with cisplatin , excluding taurine flux through unspecific leaks . Data information : Data are presented as mean ± SEM . Dashed lines in ( A - E ) represent isotonic efflux from the same individual experiments . Red arrows , change to hypotonic solution . Each panel shows the average of two independent experiments ( n = 4 for each experiments , i . e . , n = 8 in total ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-ESwelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E) −/− (C), LRRC8(B,D,E) −/− (D), or LRRC8(B,C,D) −/− (E) HEK cells compared to WT cells.FCisplatin‐induced taurine efflux (over 30 min) from WT and HAP1−/−HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.Data information: Data are presented as mean ± SEM. Dashed lines in (A-E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).


( C ) . The table summarises the number and the sex of the embryos of the indicated genotypes , recovered from timed matings of Rif1 + / - x Rif1 + / - mice , in a C57BL / 6J genetic background . The day of gestation ( E ) is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(C). The table summarises the number and the sex of the embryos of the indicated genotypes, recovered from timed matings of Rif1+/- x Rif1+/- mice, in a C57BL/6J genetic background. The day of gestation (E) is indicated.


( C ) . Time course analysis of Xist RNA expression by RT - qPCR during EB differentiation of Rif1 + / + ( Rif1 + / + + OHT ) and Rif1 - / - ( Rif1F / F + OHT ) cells at the indicated timepoints . Rif1 + / + ( solid line ) and Rif1 - / - ( dashed line ) , female ( black ) and male ( grey ) . Data are presented as mean ± standard deviation from three ( female lines ) or two ( male lines ) independent experiments . Statistical significance was determined using 2 - way ANOVA comparing female Rif1 + / + to female Rif1 - / - cell lines ( * * * * p ≤ 0 . 0001 ) . Xist RT - primers Xist ex7 F and R were used . Values are normalised to a geometric mean consisting of the expression of Gapdh , Ubiquitin and β - Actin .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin.


RIF1 association with the Xist promoter assessed by ChIP - qPCR in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female cell lines , in ESCs ( A ) P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 and 2 are two intergenic regions that serve as negative controls . Peak and cRAD represent two previously identified regions of RIF1 association ( positive control ) . See Appendix Fig . S2C for primer positions within Xist . Mean ± standard deviation from 3 independent experiments ( A ) and 2 independent experiments ( B ) . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association in Rif1 + / + cells on Xist P2 and P1 versus 5 ' . * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 and ns = not significant .

B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.


( D ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) and a wild type female mESC line ( grey ) , also harbouring one castaneus and one 129 X chromosome . Allele - specific ChIP - qPCR primers were used , cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2 . Enrichments are presented relative to input DNA . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 , * * * p ≤ 0 . 001 . Below , schematic of the Xist / Tsix alleles in the Fa2L undifferentiated cells . ( E ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) upon differentiation . The analysis was performed as in ( D ) . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 . * p ≤ 0 . 05 . Below , schematic of the Xist / Tsix alleles in the Fa2L differentiated cells . (

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O

(D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells. (E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells. (


( F ) . Quantification by ChIP - qPCR of RIF1 association with the indicated regions in the Fa2L cell line , following treatment with DMSO only ( black ) or flavopiridol ( grey ) . Primers as in ( E ) . ( G ) . Same as in ( F ) but for a wild type female mESC line . All enrichments are presented relative to input DNA . Mean ± standard deviation from three ( F ) and two ( G ) independent experiments are presented . Statistical significance was determined using Student ' s two - tailed , paired t test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E). (G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).


( A ) . Time course analysis of Xist expression by RT - qPCR during EB differentiation of female mESCs following knock down of Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Data are presented as mean ± standard deviation from three independent experiments . Statistical significance was determined using 2 - way ANOVA . Xist primers Xist ex3 F and Xist ex4 R were used . Normalisation was performed using a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . ( * p ≤ 0 . 05 ) . ( B ) . RT - qPCR analysis of Xist expression levels during differentiation of the Fa2L cells , following expression of shRNA against Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Mean ± standard deviation from a minimum of three independent experiments is presented . 2 - way ANOVA was used to determine statistical significance . ns = not significant . (

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. (


( C ) . Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase ( Control , black ) and KAP1 ( Kap1 KD , grey ) , during differentiation . Mean ± standard deviation from four independent experiments are shown . Statistical significance was determined using 2 - way ANOVA . ( * * p ≤ 0 . 01 ) . Values have first been normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.


( D ) . RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase ( control ) or against Kap1 ( Kap1 KD ) . Left : Cells with no ( 0 , red ) , one ( 1 , grey ) and two or more ( 2 , black ) Tsix foci were counted in two independent experiments , shown averaged . Statistical significance was determined by χ2 . A minimum of 110 cells were counted per each time point for each line ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Right : examples of cells with one ( top ) or two ( central and bottom ) Tsix FISH signals . Scale bars : 5μm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(D). RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase (control) or against Kap1 (Kap1 KD). Left: Cells with no (0, red), one (1, grey) and two or more (2, black) Tsix foci were counted in two independent experiments, shown averaged. Statistical significance was determined by χ2. A minimum of 110 cells were counted per each time point for each line (**p ≤ 0.01, ****p ≤ 0.0001). Right: examples of cells with one (top) or two (central and bottom) Tsix FISH signals. Scale bars: 5μm.


( A ) . ChIP - qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs ( Rif1 + / + , same as used in Fig . 3B but without OHT ) and during early differentiation . ZFP629 is a well - characterised KAP1 associated region ( positive control ) . Xite A and C indicate two regions within the Tsix enhancer Xite , Tsix region 1 indicates Tsix major promoter , Tsix region 2 indicates the Dxpas34 region , Tsix region 3 indicates a region slightly downstream of the Dxpas34 region . P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 is an intergenic region . See Appendix Fig . S2C for the positions of the primers within Xist and Tsix . The data are presented as mean ± standard deviation from three ( 2d EB and 1d EB ) and two ( ESCs ) independent experiments . Statistical significance was calculated by Student ' s two - tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB ( * p ≤ 0 . 05 and ns = not significant ) . In the inset , Tsix RNA levels were quantified by RT - qPCR during the differentiation of wild type female ESCs shown in Fig . 4F . The average of two experiments is shown . Tsix values are normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . Error bars indicate standard deviations .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.


( C ) . KAP1 association with Xist promoter in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female mESC cell lines . Ezr is an additional region known to be associated with KAP1 in mESCs . Enrichments are presented relative to input DNA . Mean ± standard deviation from a minimum of three independent experiments per cell line are displayed . Statistical significance was determined using Student ' s two - tailed , unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1 + / + versus Rif1 - / - cells ( * * p ≤ 0 . 01 ) .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).


( C ) . HEXIM1 association with the Xist promoter ( P1 and P2 ) in two independent Rif1 + / + and two Rif1 - / - mESC lines , analysed by ChIP - qPCR . Tkbp1 and Myf5 are two control regions . As in the case of KAP1 association , deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2 . Average ± standard deviation of two independent experiments . p values were calculated by two - tailed , unpaired , equal variance t - test . ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C). HEXIM1 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and two Rif1-/- mESC lines, analysed by ChIP-qPCR. Tkbp1 and Myf5 are two control regions. As in the case of KAP1 association, deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2. Average ±standard deviation of two independent experiments. p values were calculated by two-tailed, unpaired, equal variance t-test. (**p ≤ 0.01, ***p ≤ 0.001).


( E ) . Upon infection of Fa2L cells with shRNA against Kap1 or control , against Luciferase , HEXIM1 association with P2 was analysed , on both alleles , by ChIP - qPCR . As in the case of KAP1 , HEXIM1 shows preferential association with Xist P2 on the Cast allele ( future Xa , Control ) . The association is lost upon knock down of Kap1 ( Kap1 KD ) . Myf5 serves as a negative and Neat1 as a positive control regions .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(E). Upon infection of Fa2L cells with shRNA against Kap1 or control, against Luciferase, HEXIM1 association with P2 was analysed, on both alleles, by ChIP-qPCR. As in the case of KAP1, HEXIM1 shows preferential association with Xist P2 on the Cast allele (future Xa, Control). The association is lost upon knock down of Kap1 (Kap1 KD). Myf5 serves as a negative and Neat1 as a positive control regions.


( B ) Whole mount low magnification images show marking in interscale ( K10 + ) vs scale regions ,

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(B) Whole mount low magnification images show marking in interscale (K10+) vs scale regions,


C ) interscale ( K10 + ) vs scale regions , quantified in ( C ) . Error bars are SDs . The data shown is average of 4 different timepoints ( 2 week , 3month , 6month and 1 year ) each consisting of 2 mice and 5 images per mice . p - value is calculated for difference between scale vs interscale using Student ' s t - test ( mixed Anova ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) interscale (K10+) vs scale regions, quantified in (C). Error bars are SDs. The data shown is average of 4 different timepoints (2 week, 3month, 6month and 1 year) each consisting of 2 mice and 5 images per mice. p-value is calculated for difference between scale vs interscale using Student's t-test (mixed Anova).


( D ) Tail skin sections immunofluorescence stained for interscale ( K10 ) or scale ( K31 ) supra - basal markers , and basal markers . Scale bar = 20 µm or 50 µm as indicated on the panels . Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer . Dashed lines indicate ROI in basement membrane .

O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Tail skin sections immunofluorescence stained for interscale (K10) or scale (K31) supra-basal markers, and basal markers. Scale bar =20 µm or 50 µm as indicated on the panels. Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer. Dashed lines indicate ROI in basement membrane.


( E ) Quantification of images like those in ( D ) with n = 3 - 4 mice and 6 - 7 images per mouse showing preferred - gene enrichment . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effect model )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Quantification of images like those in (D) with n=3-4 mice and 6-7 images per mouse showing preferred-gene enrichment. Error bars are SDs. P-values calculated by Student's t-test (mixed effect model)


( A - F ) Human skin immunofluorescence images of mouse basal non - LRC / LRC preferred - factor staining in rete ridges ( RR ) versus inter - ridges ( IR ) . Representative images from 30 samples of different body regions and ages Scale bars are indicated on the

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Human skin immunofluorescence images of mouse basal non-LRC/LRC preferred-factor staining in rete ridges (RR) versus inter-ridges (IR). Representative images from 30 samples of different body regions and ages Scale bars are indicated on the panels


( G ) LRC / non - LRC preferred gene expression in RR and IR samples from two 30 - year - old breast samples , quantified using background subtraction P - values from paired Student ' s t - test . Error bars are SDs . 8 - 10 images were quantified each from two females .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) LRC/non-LRC preferred gene expression in RR and IR samples from two 30-year-old breast samples, quantified using background subtraction P-values from paired Student's t-test. Error bars are SDs. 8-10 images were quantified each from two females.


( A ) Schematic showing the tamoxifen injection paradigm for tdTomato - labelling of all IFE lineages in tail and back . Co - expression of markers with the lineage - traced cells at 1 - month post - tamoxifen . Dlx1 - CrER is shown as example in tail ; Scale bar = 50 µm ( B ) Quantification of images like those in ( A ) showing % tdTomato + cells expressing indicated markers . Sox6 ( black bar ) was not analyzed in K14 - Cre / EYFP lineage . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effects model ) from n = 2 - 3 mice and 5 - 6 images per replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Schematic showing the tamoxifen injection paradigm for tdTomato-labelling of all IFE lineages in tail and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER is shown as example in tail; Scale bar =50 µm (B) Quantification of images like those in (A) showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP lineage. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice and 5-6 images per replicate.


( G ) Immunofluorescence images of Sox6 staining in noUVB ( 0hr ) or UVB - exposed ( 6hrs - or 24hrs - post ) mouse back skin . ( H ) Boxplot shows % Sox6 expressing basal cells quantified from images like those in ( G ) . Central band is the mean value , the top of box indicates 25th percentile while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunofluorescence images of Sox6 staining in noUVB (0hr) or UVB-exposed (6hrs- or 24hrs-post) mouse back skin. (H) Boxplot shows % Sox6 expressing basal cells quantified from images like those in (G). Central band is the mean value, the top of box indicates 25th percentile while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


( B - G ) ( B , D , F ) Images of tail skin immune - stained for markers indicated and ( C , E , G ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-G) (B,D,F) Images of tail skin immune-stained for markers indicated and (C,E,G) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates.


( I - N ) ( I , K , M ) Images of skin immune - stained for markers indicated and ( J , L , N ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I-N) (I,K,M) Images of skin immune-stained for markers indicated and (J,L,N) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


Autophagy components promote CHIKV infection and control virus ‐ induced cell death in HeLa cells . Cells were mock infected or infected and immunoblotted for actin , p62 ( A ) or LC3 ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O

Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B).


Cells were infected for 15 h and labeled using antibodies to p62 and capsid ( C ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C).


Cells were transfected with GFP ‐ LC3 ‐ B , infected for 15 h and labeled with anti ‐ capsid antibody ( D ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D).


Mice were infected for 3 days , then whole ‐ cell lysates of muscle were immunoblotted for LC3 or actin ( E ) and muscle sections were labeled with p62 and capsid antibodies ( F ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F).


Cells were treated with DMSO ( CTRL ) or rapamycin and then infected . Viral replication ( G ) and production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I).


Cells were treated with CTRL , Beclin1 ( left ) or Atg7 ( right ) siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( K ) and production ( L ) were assessed and whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for Beclin1 , Atg7 , capsid and actin ( M ) . Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean + s . d . ( n = 3 ) . Scale bar , 10 μm . CHIKV , Chikungunya virus ; CTRL , control ; GFP , green fluorescent protein ; DMSO , dimethylsulphoxide ; siRNA , short interfering RNA .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.


Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells . Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid ( A ) , to p62 , NDP52 and capsid ( B ) , to NDP52 , LC3 ‐ C or capsid ( C ) , to p62 , ubiquitin ( FK2 ) and capsid ( D ) , or to NDP52 , ubiquitin ( FK2 ) , and capsid ( E ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O

Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E).


Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( F ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( G ) , and viral production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for p62 , capsid or actin ( I ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I).


Cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( K ) and production were assessed ( L ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L).


Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or actin ( M ) . Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean + s . d . ( n = 3 ) . Scale bar , 10 μm . C , capsid ; CHIKV , Chikungunya virus ; CTRL , control ; NDP52 , nuclear dot protein 52 kDa ; qRT ‐ PCR , quantitative reverse transcription polymerase chain reaction ; siRNA , short interfering RNA .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.


p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells . Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2 , capsid or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid ( A , B ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells. Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2, capsid or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid (A,B).


Cells were transfected with Ub ‐ HA and capsid ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ HA antibody ( C ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were transfected with Ub‐HA and capsid‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐HA antibody (C).


Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to p62 , FK2 or capsid ( D , E ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to p62, FK2 or capsid (D,E).


Cells were infected for 3 h and then transfected with empty plasmid ( CTRL ) or p62WT ‐ 3XFLAG or p62deltaUBA ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . with anti ‐ FLAG antibody . Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid ( F ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were infected for 3 h and then transfected with empty plasmid (CTRL) or p62WT‐3XFLAG or p62deltaUBA‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. with anti‐FLAG antibody. Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid (F).


Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using anti ‐ capsid antibody ( G ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using anti‐capsid antibody (G).


Cells were infected for 15 h and labeled with antibodies to p62 , LAMP ‐ 1 or capsid ( H ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

Cells were infected for 15 h and labeled with antibodies to p62, LAMP‐1 or capsid (H).


Cells were infected for 9 , 15 or 24 h and treated with DMSO ( CTRL ) or bafilomycin 3 h before the end of experiment . Whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I).


HeLa cells were transfected with empty plasmid ( CTRL ) , control protein ( cherry ‐ 3XFLAG ) or capsid ‐ 3XFLAG at fixed ( 750 ng ) or at the indicated concentrations . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( J , K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were transfected with empty plasmid (CTRL), control protein (cherry‐3XFLAG) or capsid‐3XFLAG at fixed (750 ng) or at the indicated concentrations. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (J,K).


Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( L ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (L).


Cells were infected with CHIKV for 3 h and transfected with empty plasmid ( CTRL ) , p62WT ‐ 3XFLAG , p62deltaUBA ‐ 3XFLAG or p62deltaLIR ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 24 h post transfection ( M ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were infected with CHIKV for 3 h and transfected with empty plasmid (CTRL), p62WT‐3XFLAG, p62deltaUBA‐3XFLAG or p62deltaLIR‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 24 h post transfection (M).


Human NDP52 binds nsP2 , localizes near CHIKV RCs , and restricts cell shutoff promoting cell survival in HeLa cells . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants ( top ) . Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone ( empty vector ) or to NDP52 ( bottom ) ( A ) .

O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A).


Cells were infected for 15 h , then labeled using antibodies to NDP52 and nsP2 ( B ) , or to TGN46 , nsP2 and nsP3 ( C ) .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O

Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).


Cells were treated with control ( CTRL ) ( left ) or NDP52 ( right ) siRNA , infected for 15 h , then labeled using antibodies to NDP52 , nsP2 and puromycin . Quantitative analysis was performed by counting the percentage of infected cells with TGN ‐ associated RCs ( n = 30 cells per experiment ) ( D ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D).


Cells were transfected with empty plasmid ( CTRL ) or nsP2wt ‐ 3XFLAG or nsP2R606A ‐ 3XFLAG . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( E ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E).


Cells were treated with control ( CTRL ) or NDP52 siRNA , then mock infected ( M ) or infected ( C ) for 24 h . Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52 . The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 ( F ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F).


Cells were treated with control ( CTRL ) or NDP52 siRNA , infected for 15 and 24 h and treated with puromycin . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or actin . Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin ( G ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O

Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G).


Cells were treated with CTRL or NDP52 siRNA , then transfected with NT or nsP2 ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( H ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H).


NDP52 promotes CHIKV infection in a species ‐ specific manner . MEF cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( A ) and viral production ( B ) were measured . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 ( C ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C).


MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) or hNDP52 ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ nsP2 antibody ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D).