Activation of LRRC8 channels by pro ‐ apoptotic stimuliA - CCisplatin ‐ induced iodide influx into WT ( black ■ ) , but not HAP1 − / − ( green ▼ ) HEK indicates VRAC halide current activation during apoptosis . 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 ) . difference in slopes of YFPfluorescence quenching between control cisplatin ‐ treated semiquantitatively reflects VRAC current activation . Note that increased YFP quenching with cisplatin preincubation is not due to large non ‐ specific leaks resulting from morbidity . Such leaks should lead to a fast component of YFP quenching in WT , but not HAP1 − / − after pipetting artifact that immediately follows addition of iodide ( indicated by arrows ) . DSwelling ‐ induced iodide influx into WT ( black ■ ) HAP1 − / − ( green ▼ ) HEK for comparison . Iodide ( 50 mM final ) was added in isotonic or hypotonic ( 230 mOsm final ) solution at 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 ■ ) HAP1 − / − ( green ▼ ) . Data are presented as mean ± .

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 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-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 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 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

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 two LRRC8D ‐ deficient clones ( n = 6 ) .

B-MEASURED_VAR O O O O O O 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 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 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 of indicated genotypes using 40 μM cisplatin under long ‐ term isotonic ( C ) or 200 μM cisplatin in short ‐ term hypo ‐ isotonic ( D ) conditions as function of time ( n = 3 ) . Similar results were obtained in HCT116 ( Fig EV4 ) .

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 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

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 of indicated genotypes . LRRC8 ( B , C , E ) − / − LRRC8 ( B , D , E ) − / − express only LRRC8A LRRC8D , LRRC8A 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 O O O O O O O O O O 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 (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 . number of 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

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


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 ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( for ( C , E ) compared to HAP1 − / − ) . Dotted lines in ( D ) ( F ) highlight that there is no significant difference in isotonic 60 ‐ min uptake between genotypes .

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

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 LRRC8D subunitUpper panel , example current traces ( as in Fig 3A ) of fully activated ICl , vol in HEK 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 100 mV ) of WT HEK treated with different cisplatin concentrations . No effect of 200 μM cisplatin / DMSO on ICl , vol in LRRC8D − / − HEK . Data information : Data are presented as mean ± ; 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 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-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

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 LRRC8D − / − HEK ( A , B ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 compared to WT . FCisplatin ‐ induced taurine efflux ( over 30 min ) from WT HAP1 − / − HEK 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 treated for 12 h with cisplatin , excluding taurine flux through unspecific leaks . Data information : Data are presented as mean ± . Dashed lines in ( A - E ) represent isotonic efflux from same experiments . Red arrows , change to hypotonic solution . Each panel shows 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 B-MEASURED_VAR 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 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

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 ) . table summarises number sex of of indicated genotypes , recovered from timed matings of Rif1 + / - x Rif1 + / - mice , in a C57BL / 6J genetic background . 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 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 of Xist expression by RT - qPCR during EB differentiation of Rif1 + / + ( Rif1 + / + + OHT ) Rif1 - / - ( Rif1F / F + OHT ) at indicated timepoints . Rif1 + / + ( solid ) Rif1 - / - ( dashed ) , ( black ) ( grey ) . Data are presented as mean ± standard deviation from three ( lines ) or two ( lines ) independent experiments . Statistical significance was determined using 2 - way ANOVA comparing Rif1 + / + to Rif1 - / - lines ( * * * * p ≤ 0 . 0001 ) . Xist RT - primers Xist ex7 F R were used . Values are normalised to a geometric mean consisting of expression of Gapdh , Ubiquitin β - Actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Xist promoter assessed by ChIP - qPCR in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) two Rif1 - / - ( Rif1F / F + OHT , grey ) lines , in ESCs ( A ) P1 P2 indicate two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 2 are two intergenic regions that serve as negative controls . Peak 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 ) 2 independent experiments ( B ) . p calculated by Student ' s two - , paired t comparing RIF1 association in Rif1 + / + on Xist P2 P1 versus 5 ' . * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 ns = not significant .

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

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

O O 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 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 of Xist expression by RT - qPCR during EB differentiation of mESCs following knock down of Luciferase ( Control , black ) Kap1 ( Kap1 KD , grey ) , at 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 Xist ex4 R were used . Normalisation was performed using a geometric mean consisting of expression of Rplp0 , Ubiquitin Sdha . ( * p ≤ 0 . 05 ) . ( B ) . RT - qPCR of Xist expression during differentiation of Fa2L , following expression of shRNA against Luciferase ( Control , black ) Kap1 ( Kap1 KD , grey ) , at 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in mESCs infected with shRNA directed against Luciferase ( Control , black ) 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 expression of Rplp0 , Ubiquitin Sdha .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) . FISH of Tsix expression during differentiation of mESCs expressing an shRNA directed against Luciferase ( control ) or against Kap1 ( Kap1 KD ) . Left : with no ( 0 , red ) , one ( 1 , grey ) 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 were counted per each time point for each ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Right : examples of with one ( top ) or two ( central bottom ) Tsix FISH signals . Scale bars : 5μm .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) two Rif1 - / - ( Rif1F / F + OHT , grey ) mESC lines . Ezr is an additional region known to be associated with KAP1 in mESCs . Enrichments are presented relative to input . Mean ± standard deviation from a minimum of three independent experiments per are displayed . Statistical significance was determined using Student ' s two - , unpaired t comparing KAP1 association with Xist P2 P1 in Rif1 + / + versus Rif1 - / - ( * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 Xist promoter ( P1 P2 ) in two independent Rif1 + / + two Rif1 - / - mESC lines , analysed by ChIP - qPCR . Tkbp1 Myf5 are two control regions . As in case of KAP1 association , deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 P2 . Average ± standard deviation of two independent experiments . p values were calculated by two - , unpaired , equal variance t - . ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 show marking in interscale ( K10 + ) vs scale regions ,

(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 . data shown is average of 4 different timepoints ( 2 week , 3month , 6month 1 year ) each consisting of 2 mice 5 per mice . p - value is calculated for difference between scale vs interscale using Student ' s t - ( mixed Anova ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , basal markers . Scale bar = 20 µm or 50 µm as indicated on panels . Asterisks indicate that photoshop was used to erase autoflourescent cornified layer . Dashed lines indicate ROI in basement .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) of like those in ( D ) with n = 3 - 4 mice 6 - 7 per mouse showing preferred - gene enrichment . Error bars are SDs . P - values calculated by Student ' s t - ( 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

(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 of mouse basal non - LRC / LRC preferred - factor in rete ridges ( RR ) versus inter - ridges ( IR ) . Representative from 30 samples of different regions ages Scale bars are indicated on

(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 IR samples from two 30 - year - old breast samples , quantified using background subtraction P - values from paired Student ' s t - . Error bars are SDs . 8 - 10 were quantified each from two .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 tamoxifen injection paradigm for tdTomato - labelling of IFE lineages in tail back . Co - expression of markers with lineage - traced at 1 - month post - tamoxifen . Dlx1 - CrER is shown as example in tail ; Scale bar = 50 µm ( B ) of like those in ( A ) showing % tdTomato + 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 - ( mixed effects model ) from n = 2 - 3 mice 5 - 6 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 O O O O O O O O O O O O O O O O O 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 of Sox6 in noUVB ( 0hr ) or UVB - exposed ( 6hrs - or 24hrs - post ) mouse back skin . ( H ) Boxplot shows % Sox6 expressing basal quantified from like those in ( G ) . Central band is mean value , top of box indicates 25th percentile while bottom indicates 75th percentile . Whiskers indicate minimum maximum values in data , with dots indicating potential outliers . Linear mixed model statistical was used with data from 18 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 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 ) of tail skin immune - stained for markers indicated ( C , E , G ) corresponding box plots . Central band is mean value , top of box indicates 25th percentile , while bottom indicates 75th percentile . Whiskers indicate minimum maximum values in data , with dots indicating potential outliers . Linear mixed model statistical was used with data from 18 total 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

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

(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 control ‐ induced in HeLa . were mock infected or infected 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 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).


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

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


were transfected with GFP ‐ LC3 ‐ B , infected for 15 h labeled with anti ‐ capsid 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

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 ‐ lysates of muscle were immunoblotted for LC3 or actin ( E ) muscle sections were labeled with p62 capsid antibodies ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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

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

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).


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

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

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).


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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).


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 controls induced by cytotoxic capsid in HeLa . were infected for 24 h immunoprecipitation experiments were performed using antibodies to FK2 , capsid or nonspecific IgG controls . Immunoprecipitated were revealed using antibodies to FK2 or capsid ( A , 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 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).


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

O O O O O O O O O O O O O O O O O O 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 were revealed using antibodies to p62 , FK2 or capsid ( D , E ) .

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).


were infected for 3 h 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 were revealed using antibodies to FLAG or capsid ( F ) .

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).


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 were revealed using anti ‐ capsid antibody ( G ) .

O O O O O O O O O O O O O O O O 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).


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

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


were infected for 9 , 15 or 24 h treated with DMSO ( CTRL ) or bafilomycin 3 h before end of experiment . Whole ‐ 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 B-CONTROLLED_VAR 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 were transfected with empty plasmid ( CTRL ) , control ( cherry ‐ 3XFLAG ) or capsid ‐ 3XFLAG at fixed ( 750 ng ) or at indicated concentrations . mortality ( that is , % of dead ) 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

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).


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

O O O O O O O O O O O O O O O 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).


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

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 , restricts shutoff promoting survival in HeLa . Yeast expressing Gal4 BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding Gal4 AD fused to NDP52 or indicated NDP52 deletion mutants ( top ) . Yeast expressing BD fused to nsP2 or indicated nsP2 deletion mutants were cotransformed with a plasmid encoding Gal4 AD fused alone ( empty vector ) or to NDP52 ( bottom ) ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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

O O O O 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 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).


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

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).


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

O O O O O O O O O O O O O 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).


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

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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).


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

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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).


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

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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 were treated with CTRL or NDP52 siRNA , then infected for 24 h . mortality ( that is , fold change relative to mock ‐ infected ) ( A ) viral production ( B ) were measured . Yeast expressing Gal4 BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding Gal4 AD fused to hNDP52 or mouse NDP52 ( C ) .

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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 were infected for 3 h 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 were revealed with anti ‐ nsP2 antibody ( D ) .

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).


MEF were infected for 3 h transfected with empty plasmid ( CTRL ) , hNDP52 , hLC3 ‐ C or hNDP52 hLC3 ‐ C . Viral production ( E ) mortality ( F ) were assessed 24 h post transfection . Isogenic wt or Atg5 − / − MEFs were infected for 24 h mortality ( that is , fold change relative to mock ‐ infected ) ( G ) viral production ( H ) were assessed . MEF were infected immunoprecipitation experiments were performed 24 h p . i . using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated were revealed with anti ‐ capsid antibody ( I ) .

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MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I).


MEF were treated with CTRL or p62 siRNA , then infected for 24 h . mortality ( that is , fold change relative to mock ‐ infected ) ( J ) viral production were assessed . Whole ‐ lysates of siRNA ‐ treated were immunoblotted for p62 or actin ( K ) .

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MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).


TRIP6 promotes YAP activity by inhibiting LATS1 / 2 ( A ) Full ( WT ) , - terminal half ( 1 - 277 ) , or carboxy - terminal half ( 278 - 476 ) of TRIP6 were tested for binding to LATS2 by immunoprecipitation . FLAG - TRIP6 variants were co - expressed with LATS2 - GFP in HEK293 , anti - FLAG or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes lysates were probed by western blotting for LATS2 - GFP FLAG - TRIP6 . Schematic diagram depicts TRIP6 domains ( NES : Export Signal ; LIM : LIM domain ; PDZ : PDZ domain binding motif ) .

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TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif).


( B ) Lysates from MCF10A were subjected to immunoprecipitation using anti - TRIP6 or control ( IgG ) antibodies , immune complexes lysates were probed for TRIP6 LATS1 .

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(B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1.


( C ) FLAG - TRIP6 was tested for binding to various LATS2 - GFP deletion mutants as described in part ( A ) . Schematic diagram of LATS2 shows MOB1 binding domain , autophosphorylation ( S872 ) MST1 / 2 phosphorylation sites ( T1041 ) in kinase domain .

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(C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain.


regions marked in green depict TRIP6 binding sites on LATS2 ( D ) Lysates from HEK293A transfected with control or FLAG - TRIP6 plasmid were analyzed by western blotting using indicated antibodies ( is shown in ( F ) ) .

The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)).


( E ) Lysates from control ( WT ) or CRISPR generated TRIP6 null ( TRIP6 - KO ) HEK293A were analyzed by western blotting using indicated antibodies ( shown in ( G ) ) .

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(E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)).


( F ) relative of LATS1 activating phosphorylation ( pLATS1 - 1079 , 909 ) YAP S127 inhibitory phosphorylation from ( D ) were measured relative to LATS1 YAP respectively . ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - )

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(F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test)


( G ) of LATS1 activating phosphorylation YAP inactivating phosphorylation from part ( E ) were quantified ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - ) .

(G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test).


( H ) GFP - MST2 was expressed with or without FLAG - TRIP6 in HEK293 of MST2 , MST2 activating phosphorylation ( pMST2 - T180 ) , FLAG - TRIP6 were measured by western blotting with indicated antibodies . ( Mean ± SD ; n = 3 ; n . s . ≥ 0 . 05 , T - ) .

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(H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test).


( I ) TRIP6 was overexpressed in HEK293A of TRIP6 , YAP target gene expression was analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , T - ) .

(I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test).


( J ) of YAP target gene expression was analyzed using RT - qPCR in control ( WT ) TRIP6 - KO HEK293A . ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , T - )

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(J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test)


( K ) Control ( WT ) TRIP6 - KO HEK293A were stained for YAP TRIP6 . Merged image shows YAP ( green ) , TRIP6 ( red ) , ( blue ) . of at least 100 is shown ( Mean ± SD ; n = 3 ; * * * * P ≤ 0 . 0001 , Fisher ' s ) . Scale bar = 20µm .

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(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.


( A ) LATS2 - GFP Myc - MOB1A were overexpressed in HEK293 with or without co - overexpression of full FLAG - TRIP6 FLAG - TRIP6 1 - 277 . Myc - MOB1A was immunoprecipitated using anti - Myc antibodies immune complexes were assayed for Myc - MOB1A LATS2 - GFP . of FLAG - TRIP6 , FLAG - TRIP6 1 - 277 , Myc - MOB1A , LATS2 - GFP in lysate are also shown . of LATS2 - GFP in immune complexes relative to level of Myc - MOB1A are shown in graph ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - )

(A) LATS2-GFP and Myc-MOB1A were overexpressed in HEK293 cells with or without co-overexpression of full length FLAG-TRIP6 and FLAG-TRIP6 1-277. Myc-MOB1A was immunoprecipitated using anti-Myc antibodies and immune complexes were assayed for Myc-MOB1A and LATS2-GFP levels. Levels of FLAG-TRIP6, FLAG-TRIP6 1-277, Myc-MOB1A, and LATS2-GFP in the lysate are also shown. The levels of LATS2-GFP in immune complexes relative to the level of Myc-MOB1A are shown in the graph (Mean ± SD; n=3; **P≤0.01, T-test)


( B ) Competitive binding experiments were done using purified recombinant MBP - LATS2 , GST - TRIP6 , 6His - MOB1A . MBP - LATS2 bound to maltose beads was incubated with GST - TRIP6 with or without increasing amounts of 6His - MOB1A , of each bound to MBP - LATS2 on beads at end of experiment was determined by western blotting . of input are shown ( lysate ) . binding of 6His - MOB1A GST - TRIP6 to MBP alone , use BSA as a competitor instead of 6His - MOB1A are shown as controls . numbers at bottom are referred to in text .

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(B) Competitive binding experiments were done using purified recombinant MBP-LATS2, GST-TRIP6, and 6His-MOB1A. MBP-LATS2 bound to maltose beads was incubated with GST-TRIP6 with or without increasing amounts of 6His-MOB1A, and the levels of each protein bound to MBP-LATS2 on the beads at the end of the experiment was determined by western blotting. The levels of input proteins are shown (lysate). The binding of 6His-MOB1A and GST-TRIP6 to MBP alone, and the use BSA as a competitor instead of 6His-MOB1A are shown as controls. The numbers at the bottom are referred to in the text.


( A ) MCF10A were infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 shTRIP6 - 4 ) were stained for TRIP6 LATS1 . Merged show LATS1 ( green ) , TRIP6 ( red ) ( blue ) ( of LATS1 localization at - junctions is shown in Figure EV3D ) . Scale bar = 20µm .

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(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.


( B ) LATS1 LATS2 were knocked down using siLATS1 siLATS2 SMARTPools in MCF10A . MCF10A control were treated with control siRNA ( siControl ) against fire fly luciferase . were stained for TRIP6 LATS1 as in ( A ) ( of TRIP6 localization at - junctions is shown in Figure EV3E ) . Scale bar = 20µm .

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(B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.


( C ) MCF10A were grown to high density , were stained for TRIP6 LATS1 as in ( A ) . Scale bar = 20µm .

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(C) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( D ) were grown as in ( C ) , then lysed anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes lysates were probed by western blotting for LATS1 TRIP6 . is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - ) .

(D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( E ) MCF10A grown at high density on PDMS membranes were stretched ( or not ) at 17 % elongation for 2 hours lysed while under tension . Anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes lysates were probed by western blotting for LATS1 TRIP6 . is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - ) .

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(E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( F ) were treated as in ( E ) , fixed while under tension , stained for TRIP6 LATS1 as in ( A ) . Scale bar = 20µm .

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(F) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( G ) MCF10A infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 shTRIP6 - 4 ) , were grown at high density on PDMS membranes were stretched or not ( only stretched shown ) at 17 % elongation for 2 hours , fixed while under tension , were stained for TRIP6 LATS1 as in ( A ) . Scale bar = 20µm .

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(G) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( H ) were treated as in ( G ) YAP target gene ( CTGF Cyr61 ) TRIP6 expression were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , T - ) .

(H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test).


( I ) TRIP6 expression in ( H ) in control ( shEGFP ) TRIP6 ( shTRIP6 ) knockdown just prior to stretch were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - ) .

(I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).


( A ) MCF10A were either not treated or treated separately with Latrunculin B , Blebbistatin , EGTA , serum starvation , Y27632 , were stained for TRIP6 LATS1 . Merged show LATS1 ( green ) , TRIP6 ( red ) ( blue ) . Scale bar = 20µm .

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(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) were treated as in ( A ) , then lysed anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes lysates were probed by western blotting for LATS1 TRIP6 .

(B) Cells were treated as in (A), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6.


( A ) MCF10A were lysed anti - TRIP6 or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes lysates were probed by western blotting for vinculin TRIP6 .

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(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( B ) MCF10A were either not treated ( control ) or treated separately by growth to high density , serum starvation , Latrunculin B , Blebbistatin , EGTA , or Y27632 treatment , stained using anti - vinculin antibodies by immunofluorescence . Scale bar = 20µm .

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(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.


( C ) MCF10A grown at high density on PDMS membranes were stretched ( or not ) at 17 % elongation for 2 hours fixed while under tension stained for vinculin . Scale bar = 20µm .

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(C) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm.


( D ) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A lysates were probed by western blotting for phospho - LATS1 ( T1079 S909 ) , phospho - YAP ( S127 ) , LATS1 , YAP , vinculin , tubulin antibodies relative quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - ) .

(D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( E ) Vinculin was knocked down as described in ( D ) were stained for YAP vinculin . Merged image shows YAP ( green ) , vinculin ( red ) , ( blue ) . of at least 100 is shown ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , Fisher ' s ) . Scale bar = 20µm .

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(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.


( F ) Vinculin was knocked down as described in ( D ) of vinculin YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - ) .

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(F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( G ) Vinculin was knocked down in MCF10A as described in ( D ) , grown at high density on PDMS membranes were stretched ( or not ) at 17 % elongation for 2 hours of vinculin YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - ) .

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(G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( A ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A were stained for LATS1 TRIP6 . Merged show LATS1 ( green ) , TRIP6 ( red ) ( blue ) . Scale bar = 20µm .

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(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) Vinculin was knocked down as described ( Figure 5D ) TRIP6 immune complexes lysates were probed by western blotting for LATS1 , TRIP6 , vinculin relative quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - ) .

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(B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( C ) MCF10A were subjected to different treatments as described ( Figure 4A ) TRIP6 immune complexes lysates were probed by western blotting for vinculin TRIP6 .

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(C) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( D ) of TRIP6 - vinculin binding from part ( C ) is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - ) .

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(D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( E , F ) MCF10A were treated separately ( or not ) with Latrunculin B ( E ) by serum starvation ( F ) LATS1 immune complexes lysates were probed by western blotting for vinculin , LATS1 , TRIP6 . of LATS1 - TRIP6 - vinculin binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - ) .

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(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( G ) Wild type T12 mutant of vinculin were co - expressed with LATS2 FLAG - TRIP6 anti - FLAG antibody was used to isolate immune complexes . Immune complexes lysates were probed by western blotting for vinculin , LATS2 FLAG - TRIP6 . of LATS2 - TRIP6 - vinculin ( WT or T12 ) binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - ) .

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(G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( H ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A grown at high density on PDMS membranes . membranes were stretched at 17 % elongation for 2 hours fixed while under tension stained for TRIP6 . Merged show TRIP6 ( red ) ( blue ) . Scale bar = 20µm .

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(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.


a , Survival curves of wild type ( n = 27 ) Cdc14 DKO ( n = 51 ) mice during first 6 of life . ( * * * , p < 0 . 001 ; Log - rank , Mantel - Cox ) . b , Representative of postnatal day 9 Cdc14 DKO wild - type . c , of Cdc14 DKO wild - type during first 3 of life ( n = 4 per genotype ) . Data information : Bars indicate mean ± . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction .

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a, Survival curves of wild type (n=27) and Cdc14 DKO (n=51) mice during the first 6 weeks of life. (***, p<0.001; Log-rank, Mantel-Cox test). b, Representative images of postnatal day 9 Cdc14 DKO and wild-type littermates. c, Weight of Cdc14 DKO and wild-type littermates during the first 3 weeks of life (n=4 per genotype). Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


d , Representative macroscopic pictures of brain dorsal view of wild type ( WT ) Cdc14 KO mice at postnatal 0 , 5 9 days ( P0 , P5 P9 ) . Scale bar , 2 mm . bar plots show of brain width , , respectively . Data information : Bars indicate mean ± . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show data , average from 3 WT 3 DKO mice .

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d, Representative macroscopic pictures of brain dorsal view of wild type (WT) and Cdc14 KO mice at postnatal 0, 5 and 9 days (P0, P5 and P9). Scale bar, 2 mm. The bar plots show the quantification of brain width, length and area, respectively. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


e , Representative cresyl violet of midsagittal sections of cerebellar vermis of WT Cdc14 KO mice at P0 , P5 P9 . Fissures underdeveloped in mutant samples are labeled with arrowheads . Scale bar , 700 μm . bar plots show of cerebellar vermis , number of folia average of fissures , respectively . Data information : Bars indicate mean ± . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show data , average from 3 WT 3 DKO mice .

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e, Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0, P5 and P9. Fissures underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the cerebellar vermis area, the number of folia and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


f , Detailed cresyl violet of midsagittal sections of cerebellar vermis of same samples . molecular layer ( ML ) , internal granule layer ( IGL ) external granule layer ( EGL ) are indicated . width of these layers is shown in bar plots . Data information : Bars indicate mean ± . * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show data , average from 3 WT 3 DKO mice . In f , each point represents average value of 9 different random measurements across distinctive folia for each mouse .

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f, Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML), internal granule cell layer (IGL) and external granule cell layer (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia for each mouse.


g , Representative of misdigital sections stained with DAPI ( blue ) , Calbindin ( green ) GFAP ( red ) of cerebellar vermis of wild - type ad Cdc14 DKO mice at 9 days after birth . Scale bar , 50 μm . Plots show of Purkinje dendritic ( average of 30 random measurements ) number of per 100 μm from 3 different fields from 3 different mice in each plot . Data information : Bars indicate mean ± . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction .

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g, Representative images of misdigital sections stained with DAPI (blue), Calbindin (green) and GFAP (red) of cerebellar vermis of wild-type ad Cdc14 DKO mice at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


a . Confocal microscopy of wild - type Cdc14 DKO embryonic ( ESCs ) subjected to neural differentiation . Neuronal marker TUJ1 ( green ) , glial marker GFAP ( red ) marker DAPI ( blue ) are stained quantified after 18 days in differentiation media . Each dot represents between TUJ or GFAP DAPI in an image containing at least 100 from 4 independent experiments with two different clones . * * * * p - value < 0 . 0001 . Student ' s t - . Scale bar 50 μm .

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a. Confocal microscopy analysis of wild-type and Cdc14 DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal marker TUJ1 (green), glial marker GFAP (red) and nuclear marker DAPI (blue) are stained and quantified after 18 days in differentiation media. Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.


c , Confocal microscopy of WT Cdc14 DKO ESC subjected to neural differentiation . Neural progenitor marker Nestin was stained quantified after 6 days in differentiation media . Each dot represents between Nestin DAPI in an image containing at least 100 . * * * * , p < 0 . 0001 ; Students ' t - .

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c, Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation. Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media. Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.


d , Western blot of CDC14B - GFP co - immunoprecipitations . HeLa were transfected with UTF1 GFP or CDC14B - GFP . soluble were extracted with RIPA , nucleolar - bound were extracted with high salt RIPA . Immunoprecipitations with anti - GFP antibody were performed . Total extracts immunoprecipitates were loaded UTF ( upper panel ) GFP ( lower panel ) were blotted .

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d, Western blot analysis of CDC14B-GFP co-immunoprecipitations. HeLa cells were transfected with UTF1 and GFP or CDC14B-GFP. Cytoplasmic and nuclear soluble proteins were extracted with RIPA, and nucleolar and chromatin-bound proteins were extracted with high salt RIPA. Immunoprecipitations with anti-GFP antibody were performed. Total protein extracts and immunoprecipitates were loaded and UTF (upper panel) and GFP (lower panel) were blotted.


e , Immunoprecipitation of UTF1 upon co - transfection with CDC14B . Total was extracted with high - salt RIPA immunoprecipitations with anti - UTF1 antibody were performed .

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e, Immunoprecipitation of UTF1 upon co-transfection with CDC14B. Total protein was extracted with high-salt RIPA and immunoprecipitations with anti-UTF1 antibody were performed.


f , Expression of active CDC14B , but not a phosphatase - dead ( PD ) isoform , reduces slow - mobility bands of UTF1 . Vinculin was used as a loading control .

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f, Expression of active CDC14B, but not a phosphatase-dead (PD) isoform, reduces slow-mobility bands of UTF1. Vinculin was used as a loading control.


h , In vitro kinase of recombinant human UTF1 with ERK2 , CDK1 - Cyclin B1 , CDK1 - Cyclin A2 or in absence of kinases ( C ) .

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h, In vitro kinase assay of recombinant human UTF1 with ERK2, CDK1-Cyclin B1, CDK1-Cyclin A2 or in absence of kinases (C).


j , In vitro phosphatase of UTF1 . Three different fragments of UTF1 were phosphorylated by CDK1 - Cyclin A2 subsequently exposed to wild - type or a catalytically inactive form ( PD ) of CDC14B .

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j, In vitro phosphatase assay of UTF1. Three different fragments of UTF1 were phosphorylated by CDK1-Cyclin A2 and subsequently exposed to wild-type or a catalytically inactive form (PD) of CDC14B.


a , Quantitative RT - PCR in wild - type ( WT ) or Cdc14 DKO ESCs of indicated transcripts during neural differentiation . n = 3 independent experiments ( ± S . E . M . ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Student ' s t - .

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a, Quantitative RT-PCR in wild-type (WT) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation. n=3 independent experiments (±S.E.M.); *, p<0.05; **, p<0.01; ***, p<0.001; Student's t-test.


b , Expression of a CDC14 - GFP fusion in ESCs before ( T0 ) after three days ( T3 ) in differentiation conditions . arrowheads indicate of CDC14 - GFP in nucleolus before but not after induction of differentiation . Scale bars , 5 μM . plot shows between nucleolar vs . total URF1 at T0 ( n = 26 ) or T3 ( n = 31 ) in from two separate experiments . * * * , p < 0 . 001 , Student ' s t - with Welch ' s correction .

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b, Expression of a CDC14-GFP fusion protein in ESCs before (T0) and after three days (T3) in differentiation conditions. The arrowheads indicate the concentration of CDC14-GFP in the nucleolus before but not after the induction of differentiation. Scale bars, 5 μM. The plot shows the ratio between nucleolar vs. total nuclear URF1 intensity at T0 (n= 26 cells) or T3 (n=31) in cells from two separate experiments. ***, p<0.001, Student's t-test with Welch's correction.


c , Confocal microscopy of UTF1 in WT Cdc14 DKO ESCs at time 0 ( T0 ) after 1 day ( T1 ) in differentiation media . of UTF1 mean ( FMI ) per is shown in plot to right . Figures horizontal bars indicate mean ± S . E . M . ( n = 1269 WT T0 , n = 2726 WT T1 , n = 974 Cdc14 DKO T0 , n = 1173 Cdc14 DKO T1 ) . * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Student ' s t - with Welch ' s correction .

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c, Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 (T0) and after 1 day (T1) in differentiation media. The quantification of UTF1 fluorescence mean intensity (FMI) per cell is shown in the plot to the right. Figures and horizontal bars indicate mean ± S.E.M. (n=1269 WT T0 cells, n=2726 WT T1, n=974 Cdc14 DKO T0, n=1173 Cdc14 DKO T1). **, p<0.01; ****, p<0.0001; Student's t-test with Welch's correction.


d , Western blot of UTF1 expression in WT Cdc14 DKO ESCs at time 0 after 3 days in differentiation media .

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d, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media.


e , Western blot of UTF1 expression in WT Cdc14 DKO ESCs in absence or presence of kinase inhibitors roscovitine ( R ) or trametinib ( T ) .

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e, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine (R) or trametinib (T).


f , Immunoblot of UTF1 stability after transfection of HeLa with UTF1 GFP or CDC14B - GFP . were treated with cycloheximide ( CHX ) total were detected at different time points as indicated . Cyclin B1 β - catenin are used as controls .

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f, Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B-GFP. Cells were treated with cycloheximide (CHX) and total proteins were detected at different time points as indicated. Cyclin B1 and β-catenin are used as controls.


g , Western blot of UTF1 WT or mutants in three ( UTF1 - 3KR ) or five ( UTF1 - 5KR ) lysines in absence or presence of trametinib ( T ) or roscovitine ( R ) .

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g, Western blot analysis of UTF1 WT or mutants in three (UTF1-3KR) or five (UTF1-5KR) lysines in absence or presence of trametinib (T) or roscovitine (R).


h , Immunoblot of UTF1 mutants in SPOP , SIAH or SPOP + SIAH potential binding sites , as well as S48D S54D phosphorylation sites . Actin was used as a loading control .

h, Immunoblot of UTF1 mutants in SPOP, SIAH or SPOP+SIAH potential binding sites, as well as S48D and S54D phosphorylation sites. Actin was used as a loading control.


A . Fura2 340 / 380 time traces from M - CSF - differentiated macrophages derived from conditionally - immortalised macrophage precursor lines ( M - MØP ) of Plcg2P522 mice ( P522 , blue ) Plcg2R522 mice ( R522 , red ) . One set of lines , generated from mice , is shown . were exposed to 5µg / ml anti - FcγRII / III along with 20µM EGTA 2µM Ionomycin as indicated . Data shows mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc ( * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ) . B . Fura2 340 / 380 time traces from microglia derived from cortex of Plcg2R522 mice ( blue : P522 ) Plcg2R522 mice ( red : R522 ) with or without pre - exposure for 2 hours with Edelfosine ( 10µM ) . were exposed to 5µg / ml anti - FcγRII / III along with EGTA 2µM Ionomycin . Data shows mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc ( * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ) . C

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A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C


C - E . M - MOPS ( C ) , mouse microglia ( D ) hIPSC ( E ) ( blue : P522 red : R522 ) were loaded with Fluo - 8 Ca2 + indicator examined for peak changes in fluoresce after exposure to DOPS - liposomes ( 25 μg / ml ) . Data information : Data shown as mean ± SD of 3 independent experiments . data were analysed by two - way ANOVA with Sidak ' s post - ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 ) .

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C-E. M-MOPS (C), primary mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001).