Figure 3 ( A ) HH / SMO promotes slow - migrating forms of Smaug . Western blot analysis of Cl8 cells that transiently express HA - SmaugWT alone , or together with either SMOWT - GFP or SMOPKA - SD - GFP , in the presence or in absence of HH . GMAP serves as a loading control . Here and in the other panels , the antibodies used are indicated on the left . Here and in ( B , C ) , the black arrows indicate the unphosphorylated form of HA - SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA - SmaugWT . NT : not transfected . See a phosphatase assay in Fig EV4A . ( B ) Smaug phosphorylation requires its interaction with SMO . Western blot analysis of Cl8 cells expressing HA - SmaugWT , in presence of HH , in combination with SMO ∆ 958 - GFP , SMOPKA - SD - GFP or SMOPKA - SD , ∆ 978 - GFP , as indicated . ( C ) FU controls Smaug phosphorylation . Extracts of Cl8 cells expressing SNAP - Smaug with SMOPKA - SD FU - SD - GFP ( with HH ) in combination with GFP - FUWT , GAP - GFP - FUWT , Myc - FUEE or GAP - null - FUDANA ( as indicated ) were labeled for the SNAP tag before electrophoresis . After quantification of the gels , the percentage ( % ) of phosphorylated Smaug ( % of phosphorylated forms of SNAP - Smaug total amounts of SNAP - Smaug ) was estimated . In C to E , the mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom , and the statistical analysis was done by one tailed bivariate Wilcoxon rank test . See also fig EV4C , D . ( D ) Phosphorylation of Smaug by activated FU requires the co - expression of SMO . Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN - SNAP - Smaug with GAP - GFP - FUWT and SMOWT alone or together ( as indicated ) N = 3 ( billogical triplicates ) . Note that the effect of GAP - GFP - FUWT and SMOWT together is lower than the one observed with GAP - GFP - FUWT and SMOPKA - SD FU - SD - GFP together ( in panel C ) . This could have multiple nonexclusive causes , including the fact that SMOWT is present at much lower levels than SMOPKA - SD FU - SD [ 38 ] and / or the involvement of another kinase . See also Fig EV4B . Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2 . ( E ) Forcing the interaction of Smaug with activated FU leads to a SMO - independent phosphorylation of Smaug . Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN - SNAP - Smaug with GFP - FU , FU - SBR , FUEE - SBR or FUDANA - SBR , with or without HH , as indicated . N = 3 ( biological triplicates ) . See also Fig EV4C and D . Note that the levels of Smaug phosphorylation observed in presence of FUDANA - SBR are not lower than those seen with GFP - FU . This suggests an incomplete inhibition of endogenous FU , which may be due to the trapping of FUDANA - SBR by Smaug . See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.(B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.


Figure 4 ( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The % of phosphorylated SNAP - Smaug protein ( determined as in Fig 3 ) ( A ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control . ( B ) Effect of FUEE - SBR on Smaug activity . The relative levels of reporter expression obtained in the same experiment as in ( A - C ) are represented as a function of the % of phosphorylated Smaug . Color of data points correspond to ng of transfected FUEE - SBR in A - C . ( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The relative levels of of λN - SNAP - Smaug protein ( C ) ( determined as in Fig 2 ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control . ( D ) Effect of FUEE - SBR on Smaug subcellular localization . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT ( 1 ) , FUEE - SBR - mCherry ( 2 ) , FUDANA - SBR - mCh ( 3 ) or expressing GFP - SmaugWT together with FUEE - SBR - mCherry ( 4 - 4 ' ' ) or GFP - SmaugWT together with FUDANA - SBR - mCh ( 5 - 5 ' ' ) . The merge images in 4 " and 5 " show GFP - SmaugWT in green and FUEE - SBR - mCh or FUDANA - SBR - mCh in red . ( 6 ) In presence of FUEE - SBR , 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci . In presence of FUDANA - SBR , 100 % of the cotransfected cells ( n = 241 ) show Smaug and FU distributed in bodies and colocalized . 250 ng of plasmid were used for each construct . Scale bar ( shown in D1 , identical for all panels ) :

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ


Figure 5 ( A ) Effect of FUEE - SBR and HH / SMO on endogeneous Smaug target mRNAs in Cl8 cells . Relative mRNA levels estimated using semi - quantitative RT - PCR for various Smaug ' s targets in Cl8 cells expressing FUEE - SBR ( black ) , FUEE ( dark grey ) , or FUDANA - SBR ( light grey ) were reported with respect to levels of a GFP control . mRNA levels were set to 1 for the GFP control . The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3 . See also Fig EV5E . Here and in Fig EV5D : RNA encoding mitochondrial proteins are written in blue . N = 3 ( biological triplicates ) and the quantifications were repeated 3 times on each sample . The results are presented as the means ± standard error . The differences between groups were assessed using Student ' s t - tests with Prism V8 . 2 . 1 software . A p value < 0 . 05 was considered to indicate a statistically significant difference ( B ) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc . Relative levels of Smaug ' s targets mRNAs in MS1096 ; UAS - hh or hhts and their respective controls ( as in Fig EV5X ) . N = 3 , with xx disc for each phenotype . ( C ) Effect of smaug mutation on [ fu1 ] wing class distribution . Percent distribution of phenotypic classes ( defined in Appendix Figure S4 ) in fu1 males in presence of zero ( fu1 ) , one ( fu1 ; ; smaug47 / + ) or two copies ( fu1 ; ; smaug47 / smaug47 ) of the smaug47 allele . The wings were double - blindly classified in " weak " and " strong " according to the LV3 - V4 defects ( see Fig EV6B ) . * Chi2 statistical analysis gave a p of 0 , 015 for fu1 and fu1 ; ; smaug47 / + distributions and of 0 , 0024 when comparing fu1 ; ; smaug47 / + to fu1 ; ; smaug47 / smaug47 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.


Figure 1C ) MiNA analysis of HeLa cells stained with the membrane potential - independent dye MitoTracker ™ Green ( MTG , 100 nM ) to visualize and determine the mitochondrial area ( = mitochondrial footprint ) . Left : HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Co - localization shown in yellow . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . D ) Left : HeLa IF1 - KO cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . E ) Left : HeLa IF1 - H49K cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . Co - localization shown in yellow . Scale bars = 10 µm . F )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)


Figure 2A ) BN - PAGE and subsequent immuno - blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1 . Monomers ( M ) , dimers ( D ) and oligomers ( O ) were detected . Left panel : Coomassie - stained gel . Right panels : Immunoblotting and detection of ATP synthase and IF1 . WT : wild type HeLa cells ; IF * : cells expressing IF1 - H49K . Stars indicate oligomeric IF1 forms . I : complex I ; III : complex III . D ) Assembly of sEcGFP - tagged subunits CoxVIIIa - sEcGFP ( CIV # ) into complex IV , and assembly of subunits γ - sEcGFP ( SUγ # ) and SU e - sEcGFP ( Sue # ) into ATP synthase . Proteins were separated by BN - PAGE and immunoblotted . Lanes # 1 - # 10 were cut into three pieces for further evaluation . Lanes 1 - 2 : complex IV assembly in WT and CoxVIIIa - sEcGFP - expressing cells , detection of CIV with anti - Cox1 . Lanes 3 - 4 : complex V assembly in WT and SUγ - sEcGFP - expressing cells , detection with anti - SU γ . Lanes 8 - 10 : assembly of CIV # into CIV , SU γ - sEcGFP ( SUγ # ) into CV , and SU e - sEcGFP ( SUe # ) into CV . At lane # 6 in the Coomassie - stained gel , the positions of molecular weight markers ( M ) are shown . SC : respiratory supercomplexes ; CIV2 : complex IV dimers ; CIV : complex IV monomer ; s1 : F1 - c8 subcomplexes ; s2 : subcomplexes of CV containing subunits b , e and g He et al . , 2018 ( ) . Blue * * : dimeric F1F0 ATP synthase ; blue * : monomeric F1F0 ATP synthase . Red asterisks : respiratory

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes


Figure 3A , A ' Trajectory maps of ATP synthase labeled at SU γ ( A ) and SU g ( A ' ) in single mitochondria . Insets : Detailed views of the diffusion of CV - SU γ ( A ) and CV - SU g ( A ' ) . Scale bars : 1 μm overview and 0 . 5 μm insets .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets.


Figure 4B ) Diffusion coefficients for ATP synthase under control conditions ( 5 . 6 mM glucose ) , and under OXPHOS conditions . The probability density function ( PDF ) and cumulative density function ( CDF ) of - log D is plotted . Number of trajectories analyzed : 8622 ( Glc ) and 10333 ( Gal ) from mitochondria in at least 5 different cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells.


Figure 5C ) Images showing matrix pH equilibration with extracellular pH . Shown are ratiometric fluorescence images of MPP - sEcGFP before and after addition of the uncouplers and inhibitors FCCP , nigericin and oligomycin ( plus 0 . 002 % w / v Triton X - 100 where indicated ) . False color images ( λ511 / λ464 ) indicate ratio values . D ) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH = 8 , pH = 7 . 4 and pH = 6 . 5 . Scale bars = 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E


Figure 6B ) Mitochondrial pH values at SU e , SU γ and in the matrix bulk in hyper - glycolytic cells ( high glucose : HGlc , 25 mM ) . C ) pH at SU e , SU γ and in the matrix in respiring cells ( Gal , 10 mM ) . D ) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM). C) pH at SU e , SU γ and in the matrix in respiring cells (Gal, 10 mM). D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain. Da


Figure 7A ) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic ( HGlc ) and respiring ( Gal ) conditions . Where indicated , 5 µg / mL oligomycin was added to inhibit CV activity . B ) Mitochondrial pH at CV SU e in IF1 - KO cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . C ) Mitochondrial pH at CV SU e in IF1 - OE cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . D ) F ) Effect of inhibiting the respiratory chain at complex III ( CIII ) with antimycin A ( AA ) on the local pH at SU e in WT cells ( glycolytic conditions ) . G ) Local pH at SU e in IF1 - KO cells after inhibiting CIII . H ) Local pH at SU e in IF1 - OE cells after inhibiting CIII .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D)F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da


Figure 8A ) Mitochondrial pH at CV SU e is compared for HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions . D ) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions . J ) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ . The pH was color coded according to respective emission ratio . The pH at SU γ in IF1 - H49K - expressing cells is compared with WT cells . ( Scale bar : 1 µm ) .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm).


Figure 9A ) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1 . Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) were determined with an automatic flux analyzer ( Seahorse / Agilent ) . After monitoring basal respiration , oligomycin ( 1 µM ) , and rotenone plus antimycin A ( Rot ; 0 . 5 µM ; AA , 0 . 5 µM ) were added sequentially to determine glycolytic and mitochondrial ATP production rates . ~ 12 technical replicates per measurement , N = 4 independent assays .

Figure 9A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.


Figure 1A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP - positive cells was determined 16 h later by flow cytometry . B LDH release from HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . At 24 h post transfection , cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined . Graphs show mean and s . d . of quadruplicate wells . C - D . PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD in the presence of osmoprotectants . At 24 h post transfection , PEGs of the indicated molecular weights were added to a final concentration of 30 mM , cells were treated with 250 ng / ml doxycycline for 8 h and the level of PI staining ( C ) or LDH release ( D ) was determined . E - F . PI staining of and LDH release from LPS - primed primary BMDMs infected with log - phase S . typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight ( numbers on the X - axis , 30 mM final concentration ) . * p < 0 . 05 as determined by student ' s t test . Graphs show mean and s . d . of quadruplicate wells . Data are representative of at least three independent expeirments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.


Figure 2A . Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD , cleaved GSDMD and α - tubulin in the cell lysates of immortalized LPS - primed WT macrophages left uninfected ( NS ) or infected for 10 - 40 minutes with log phase S . typhimurium ( MOI = 50 ) . C . Fractionation and immunoblot analysis for GSDMD , Na + K + ATPase , VDAC ( Voltage - dependent anion channel ) , HDAC1 ( Histone deacetylase 1 ) and GAPDH ( Glycerinaldehyd - 3 - phosphat - Dehydrogenase ) of WT macrophages infected for 10 and 20 minutes with log phase S . typhimurium ( MOI = 50 ) . Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane . D . Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S . typhimurium ( MOI = 50 ) . Extraction was carried in variable conditions as described in the Material and Methods section .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane.D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.


Figure 3A . Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . The protein was cleaved in a time - dependent manner into two bands of 31 kDa ( GSDMDNterm ) and 22 kDa ( GSDMDCterm ) . B . Cross - linking experiment of full - length and cleaved GSDMD . GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . After enzymatic cleavage , GSDMDNterm is highly cross - linked by DSS , resulting in the gel - impenetrating species highlighted by the arrow . GSDMDCterm is not cross - linked . C . GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase - 1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E . coli . After 2 h , the lipid fraction ( L ) was separated from the supernatant ( S ) by ultracentrifugation at 4C for 1 h at 120 , 000g .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm).B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked.C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.


Figure 4A - I . Dye release time courses from liposomes as a percentage of maximal release . A . Five different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli polar lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 130 , 100 , 65 ( colored dark to light orange ) . The time point of 20 min is highlighted by a vertical dashed line . A - I . Dye release time courses from liposomes as a percentage of maximal release . B . Five different reactions , where 130 nM of GSDMD and 400μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli extract polar lipid were incubated with caspase - 1 concentration of ( nM ) : 15 , 8 , 5 , 2 . 5 , 1 . 2 ( colored dark to light blue ) . The time point of 20 min is highlighted by a vertical dashed line . A - I . Dye release time courses from liposomes as a percentage of maximal release . C . Three different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 100 ( colored dark to light green ) . A - I . Dye release time courses from liposomes as a percentage of maximal release . D . Dye release at 20 minutes reaction as a function of GSDMD ( dark orange ) and caspase - 1 ( dark blue ) concentrations . Errors bars for three independent experiments are shown . A - I . Dye release time courses from liposomes as a percentage of maximal release . E . Two different sets of reactions , where wild type GSDMD ( dark to light orange ) and the mutant GSDMDI104N ( dark to light blue ) were independently incubated at the concentrations of 260 , 130 and 65 nM with 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes . The time point of 60 minutes is highlighted by a vertical dashed line . A - I . Dye release time courses from liposomes as a percentage of maximal release . F . Dye release at 60 minutes of reaction as a function of GSDMD wild type ( dark orange ) and GSDMDI104N ( dark blue ) concentration . Error bars for three independent experiments are shown . A - I . Dye release time courses from liposomes as a percentage of maximal release . G - I . Dye release from 400 μM liposomes loaded with the 6 - carboxifluorescein - derivates FD - 20 , FD - 40 and FD - 150 , with variable Stokes diameters , as indicated . 130 nM of GSDMD and 5 nM caspase - 1 were incubated with the liposomes .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line.A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line.A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green).A-I. Dye release time courses from liposomes as a percentage of maximal release. D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown.A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line.A-I. Dye release time courses from liposomes as a percentage of maximal release. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown.A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.


Figure 5A - C . Cryo - EM micrographs of GSDMDNterm pores in E . coli extract polar lipids liposomes . The micrographs were acquired at protein / lipid molar ratios of 1 / 1000 , 1 / 500 and 1 / 100 , respectively . Black arrows indicate ring - shaped structures corresponding to oligomeric GSDMDNterm pore forms . Scale bars = 80 nm . D . Proteoliposome with protein / lipid molar ratio of 1 / 100 at higher magnification . Scale bar = 80 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm.D. Proteoliposome with protein/lipid molar ratio of 1/100 at higher magnification. Scale bar = 80 nm.


Figure 6A . AFM topograph of GSDMDNterm bound to lipid membranes of E . coli extract polar lipids . Overview topograph showing arc - , slit - and ring - like GSDMD oligomers . B - E . High - resolution AFM topographs and height profiles of GSDMDNterm oligomers ( red curves ) . Height profiles were measured along the red lines in the topographs . Dashed zero lines ( 0 nm ) indicate the surface of the lipid membrane ; heights of < − 4 nm indicate that the oligomers formed transmembrane pores . Topographs were taken in buffer solution ( 150 NaCl , 20 mM Hepes , pH 7 . 8 ) at room temperature . The experiment was reproduced at least three times using independent liposome and GSDMD preparations . F - H . Analysis of GSDMDNterm oligomers bound to lipid membranes of E . coli extract polar lipids . F . GSDMDNterm oligomers assembled into arcs , slits and rings leading to transmembrane ( TM ) pores or not transmembrane aggregate ( n = 218 ) . G . Height of GSDMDNterm oligomers protruding from the lipid membrane . Bars represent mean and error bars represent s . e . m . ( n = 218 ) . H . Distribution of the diameters of rings formed by GSDMDNterm oligomers . The average distribution was 21 . 2 ± 5 . 6 nm ( n = 164 ; average ± s . d . ) and the bin size was 3 nm .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers.B-E. High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves). Height profiles were measured along the red lines in the topographs. Dashed zero lines (0 nm) indicate the surface of the lipid membrane; heights of <−4 nm indicate that the oligomers formed transmembrane pores. Topographs were taken in buffer solution (150 NaCl, 20 mM Hepes, pH 7.8) at room temperature. The experiment was reproduced at least three times using independent liposome and GSDMD preparations.F-H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.


Figure 1 ( E ) Expression levels of LSD1 in MGC - 803 , MKN - 45 , HGC - 27 , BGC - 823 , and NCI - N87 cell lines and their corresponding sEVs . The samples with equal amounts of proteins were loaded ( n = 3 biological replicates ; mean ± SEM ; compared with the NCI - N87 ; * P = 0 . 0432 ( cell / MGC - 803 ) , * P = 0 . 0300 ( cell / HGC - 27 ) , * * P = 0 . 0306 ( cell / BGC - 823 ) , * * P = 0 . 0018 ( sEVs / MGC - 803 ) , * * P = 0 . 0029 ( sEVs / HGC - 27 ) , and * * * P = 0 . 0003 ( sEVs / BGC - 823 ) ; two - tailed unpaired Student ' s t - test ; GAPDH was used as a loading control for cell lysis ; CD9 was used as a loading control for sEV lysis ) . ( J ) Expression levels of LSD1 , CD63 , and CD9 in sEVs with indicated treatment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 1(E) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *P = 0.0432 (cell/MGC-803), *P = 0.0300 (cell/HGC-27), **P = 0.0306 (cell/BGC-823), **P = 0.0018 (sEVs/MGC-803), **P = 0.0029 (sEVs/HGC-27), and ***P = 0.0003 (sEVs/BGC-823); two-tailed unpaired Student's t-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis).(J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.


Figure 2 ( A ) Confocal microscopy image analysis of sEV fusion to MGC - 803 cells . The MGC - 803 ( left side ) and MKN - 45 ( right side ) cells were treated with sEVs derived from MGC - 803 cells ( Con sEVs ) or LSD1 knockout ( KO ) MGC - 803 cells ( KO sEVs ) and stained with PKH26 for 12 h . Additionally , the cell membrane was stained with Dio , while the nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar = 100 µm . ( B ) Immunofluorescence confocal microscopy analysis of LSD1 ( green ) in LSD1 KO MGC - 803 cells ( left panel ) and LSD1 KO MKN - 45 cells ( right panel ) incubated with 20 μg / mL Con sEVs and KO sEVs for 12 h . The cell membrane was stained with PKH26 , while the nuclei were stained with DAPI . Scale bar = 100 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.


Figure 3 ( F ) Expression levels of LSD1 , Nanog , OCT4 , SOX2 , and CD44 in LSD1 KO MGC - 803 cells . ( N ) Immunoprecipitation of Kme1 / 2 on SOX2 ( WT indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - WT ; Mut indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - Mut ; WT , wild type ; Mut , mutant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(F) Expression levels of LSD1, Nanog, OCT4, SOX2, and CD44 in LSD1 KO MGC-803 cells.(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).


Figure 4 ( C ) Expression levels of LSD1 , OCT4 , and SOX2 in MGC - 803 and MKN - 45 cells incubated with 20 μg / mL sEVs from MGC - 803 or LSD1 KO MGC - 803 cells for 48 h . ( D ) Quantification of the results of ( C ) ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0385 ( LSD1 ) , * P = 0 . 0160 ( OCT4 ) , and * * * P = 0 . 0006 ( SOX2 ) for MGC - 803 ; * P = 0 . 0299 ( LSD1 ) , * P = 0 . 0147 ( OCT4 ) , and * P = 0 . 0258 ( SOX2 ) for MKN - 45 ; two - tailed unpaired Student ' s t - test ) . ( F ) Sphere formation assay results of MGC - 803 ( upper panel ) and MKN - 45 ( bottom panel ) cells incubated with 20 μg / mL sEVs from HEK293T cells ( left panel ) , WT - LSD1 sEVs ( middle panel ) , and LSD1 K661A sEVs ( right panel ) for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0021 ( MGC - 803 ) and * * P = 0 . 0020 ( MKN - 45 ) ; two - tailed unpaired Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h. (D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *P = 0.0385 (LSD1), *P = 0.0160 (OCT4), and ***P = 0.0006 (SOX2) for MGC-803; *P = 0.0299 (LSD1), *P = 0.0147 (OCT4), and *P = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's t-test). (F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0021 (MGC-803) and **P = 0.0020 (MKN-45); two-tailed unpaired Student's t-test).


Figure 5 ( F ) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay . Scale bar = 50 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0024 , * * P = 0 . 0088 , and * P = 0 . 0342 ; two - tailed unpaired Student ' s t - test ) . ( I and J ) Expression levels of LSD1 in sEVs isolated from the plasma . CD9 and CD63 were used as markers of sEVs . CD9 was used as a loading control for sEV lysis ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0256 ( patient 1 ) , * * P = 0 . 0068 ( patient 2 ) , * * P = 0 . 0013 ( patient 3 ) , * * P = 0 . 0031 ( patient 4 ) , * * * P = 0 . 0009 ( patient 5 ) , * * P = 0 . 0012 ( patient 6 ) , * * * P = 0 . 0007 ( patient 7 ) , * * * P = 0 . 0010 ( patient 8 ) , * * * P = 0 . 0001 ( patient 9 ) , and * P = 0 . 0461 ( patient 10 ) ; two - tailed unpaired Student ' s t - test ) . ( K ) Sphere formation assay results of MGC - 803 cells treated with sEVs as indicated for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0019 and * * P = 0 . 0050 ; two - tailed unpaired Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *P = 0.0256 (patient 1), **P = 0.0068 (patient 2), **P = 0.0013 (patient 3), **P = 0.0031 (patient 4), ***P = 0.0009 (patient 5), **P = 0.0012 (patient 6), ***P = 0.0007 (patient 7), ***P = 0.0010 (patient 8), ***P = 0.0001 (patient 9), and *P = 0.0461 (patient 10); two-tailed unpaired Student's t-test).(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).


Figure 6Proliferation assay results of MGC - 803 ( A ) , MKN - 45 ( B ) , NCI - N87 ( C ) cells treated with oxaliplatin along with indicated treatments ( n = 3 biological replicates , mean ± standard error of mean ( SEM ) ; * P = 0 . 0204 ( MGC - 803 ) , * P = 0 . 0498 ( MKN - 45 ) , * P = 0 . 0473 ( NCI - N87 ) , and * P = 0 . 0019 ( LSD1 KO MGC - 803 ) ; two - tailed unpaired Student ' s t - test ) . Representative images of tumors ( E ) and the tumor weight ( F ) of mice treated with oxaliplatin in the presence or absence of sEVs ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0488 ; two - tailed unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).


Figure 1Loss of LRRC8D causes resistance to carboplatin and cisplatin , but not to oxaliplatin . Survival of parental , vector ‐ transduced , or LRRC8D ‐ deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin , carboplatin , and oxaliplatin . The corresponding IC50 values and 95 % confidence interval ( CI ) are given in Appendix Table S2 . Data are presented as mean ± SEM .

Figure 1Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.


Figure 3A , BVRAC currents ( ICl , vol ) of the HAP1 ( A ) and KBM7 ( B ) haploid cell lines . Left panels , example current traces of ICl , vol fully activated by hypotonic cell swelling measured with the voltage ‐ clamp protocol shown in ( A ) . Dashed lines indicate zero current . Right panels , averaged current / voltage relationships of maximally activated ICl , vol . Consistent with VRAC currents , they needed hypotonic swelling for activation , displayed an I − > Cl − permeability sequence , and were blocked by DCPIB ( Appendix Fig S2A - H ) . The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E ( Fig EV1 ) which accelerates VRAC inactivation ( Voss et al , 2014 ) . At potentials > + 100 mV , also KBM7 currents inactivated ( Appendix Fig S2I ) . Data are presented as mean ± SEM ; n = 5 - 10 . CCDependence of regulatory volume decrease ( RVD ) of HEK cells on LRRC8 genes . Cells were exposed to hypotonic medium starting at t = 0 , and intracellular calcein fluorescence was followed over ˜ 1 h as semiquantitative measure of cell volume . Data are presented as mean values ± SEM from sixteen wells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, BVRAC currents (ICl,vol) of the HAP1 (A) and KBM7 (B) haploid cell lines. Left panels, example current traces of ICl,vol fully activated by hypotonic cell swelling measured with the voltage‐clamp protocol shown in (A). Dashed lines indicate zero current. Right panels, averaged current/voltage relationships of maximally activated ICl,vol. Consistent with VRAC currents, they needed hypotonic swelling for activation, displayed an I− > Cl− permeability sequence, and were blocked by DCPIB (Appendix Fig S2A-H). The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E (Fig EV1) which accelerates VRAC inactivation (Voss et al, 2014). At potentials > +100 mV, also KBM7 currents inactivated (Appendix Fig S2I). Data are presented as mean ± SEM; n = 5-10.CCDependence of regulatory volume decrease (RVD) of HEK cells on LRRC8 genes. Cells were exposed to hypotonic medium starting at t = 0, and intracellular calcein fluorescence was followed over ˜1 h as semiquantitative measure of cell volume. Data are presented as mean values ± SEM from sixteen wells.


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Activation 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.


Figure 6Carboplatin uptake into control KBM7 cells and two LRRC8D ‐ deficient clones ( n = 6 ) . Hypotonicity ‐ stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone ( CBX ) , a non ‐ specific blocker of VRAC ( n = 3 ) . Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long ‐ term isotonic ( C ) or 200 μM cisplatin in short ‐ term hypo ‐ and isotonic ( D ) conditions as function of time ( n = 3 ) . Similar results were obtained in HCT116 cells ( Fig EV4 ) . Cisplatin uptake ( 200 μM ) into HEK cells of indicated genotypes . LRRC8 ( B , C , E ) − / − and LRRC8 ( B , D , E ) − / − cells express only LRRC8A and LRRC8D , and LRRC8A and LRRC8C , respectively ( n = 3 for WT , 6 for LRRC8D − / − , 9 for LRRC8A − / − in E ; n = 3 in F ) . Mean current densities of maximally activated ICl , vol at − 80 mV . The number of cells is indicated for each column . Ratio of LRRC8 ‐ dependent swelling ‐ activated cisplatin uptake ( 60 min ) to mean ICl , vol ( as in G ) as function of genotype . Data information : Data are presented as mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 ( for ( C , E ) compared to HAP1 − / − cells ) . Dotted lines in ( D ) and ( F ) highlight that there is no significant difference in isotonic 60 ‐ min uptake between the genotypes .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F).Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column.Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.


Figure 7Cisplatin - DMSO inhibition of ICl , vol depends on the LRRC8D subunitUpper panel , example current traces ( as in Fig 3A ) of fully activated ICl , vol in HEK cells exposed to hypotonic solution containing vehicle ( 0 . 3 % DMSO ) or 200 μM cisplatin in 0 . 3 % DMSO . Dashed lines indicate zero current . Lower panel , ICl , vol current densities ( at − 100 mV and 100 mV ) of WT HEK cells treated with different cisplatin concentrations . No effect of 200 μM cisplatin / DMSO on ICl , vol in LRRC8D − / − HEK cells . Data information : Data are presented as mean ± SEM ; the number of experiments is given for each bar ; * P < 0 . 05 ; * * P < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Cisplatin-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.


Figure 8Swelling ‐ induced efflux of 3 [ H ] ‐ taurine from WT and LRRC8D − / − HEK cells ( A , B ) and partial rescue by transient transfection of LRRC8D ( B ) . Rescue is incomplete due to low transfection / expression efficiency of LRRC8D ( Voss et al , 2014 ) . C - ESwelling ‐ induced efflux of 3 [ H ] ‐ taurine from LRRC8 ( B , C , E ) − / − ( C ) , LRRC8 ( B , D , E ) − / − ( D ) , or LRRC8 ( B , C , D ) − / − ( E ) HEK cells compared to WT cells . FCisplatin ‐ induced taurine efflux ( over 30 min ) from WT and HAP1 − / − HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h , or without cisplatin ( 0 h ) . Carbenoxolone ( CBX ; 100 μM ) blocks taurine efflux from WT cells treated for 12 h with cisplatin , excluding taurine flux through unspecific leaks . Data information : Data are presented as mean ± SEM . Dashed lines in ( A - E ) represent isotonic efflux from the same individual experiments . Red arrows , change to hypotonic solution . Each panel shows the average of two independent experiments ( n = 4 for each experiments , i . e . , n = 8 in total ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D −/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).C-ESwelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E) −/− (C), LRRC8(B,D,E) −/− (D), or LRRC8(B,C,D) −/− (E) HEK cells compared to WT cells.FCisplatin‐induced taurine efflux (over 30 min) from WT and HAP1−/−HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.Data information: Data are presented as mean ± SEM. Dashed lines in (A-E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).


Figure 1 ( C ) . The table summarises the number and the sex of the embryos of the indicated genotypes , recovered from timed matings of Rif1 + / - x Rif1 + / - mice , in a C57BL / 6J genetic background . The day of gestation ( E ) is indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3RIF1 association with the Xist promoter assessed by ChIP - qPCR in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female cell lines , in ESCs ( A ) P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 and 2 are two intergenic regions that serve as negative controls . Peak and cRAD represent two previously identified regions of RIF1 association ( positive control ) . See Appendix Fig . S2C for primer positions within Xist . Mean ± standard deviation from 3 independent experiments ( A ) and 2 independent experiments ( B ) . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association in Rif1 + / + cells on Xist P2 and P1 versus 5 ' . * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 and ns = not significant . ( D ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) and a wild type female mESC line ( grey ) , also harbouring one castaneus and one 129 X chromosome . Allele - specific ChIP - qPCR primers were used , cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2 . Enrichments are presented relative to input DNA . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 , * * * p ≤ 0 . 001 . Below , schematic of the Xist / Tsix alleles in the Fa2L undifferentiated cells . ( E ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) upon differentiation . The analysis was performed as in ( D ) . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 . * p ≤ 0 . 05 . Below , schematic of the Xist / Tsix alleles in the Fa2L differentiated cells . ( ( F ) . Quantification by ChIP - qPCR of RIF1 association with the indicated regions in the Fa2L cell line , following treatment with DMSO only ( black ) or flavopiridol ( grey ) . Primers as in ( E ) . ( G ) . Same as in ( F ) but for a wild type female mESC line . All enrichments are presented relative to input DNA . Mean ± standard deviation from three ( F ) and two ( G ) independent experiments are presented . Statistical significance was determined using Student ' s two - tailed , paired t test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.(D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells. (E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells. ((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).


Figure 4 ( A ) . Time course analysis of Xist expression by RT - qPCR during EB differentiation of female mESCs following knock down of Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Data are presented as mean ± standard deviation from three independent experiments . Statistical significance was determined using 2 - way ANOVA . Xist primers Xist ex3 F and Xist ex4 R were used . Normalisation was performed using a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . ( * p ≤ 0 . 05 ) . ( B ) . RT - qPCR analysis of Xist expression levels during differentiation of the Fa2L cells , following expression of shRNA against Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Mean ± standard deviation from a minimum of three independent experiments is presented . 2 - way ANOVA was used to determine statistical significance . ns = not significant . ( ( C ) . Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase ( Control , black ) and KAP1 ( Kap1 KD , grey ) , during differentiation . Mean ± standard deviation from four independent experiments are shown . Statistical significance was determined using 2 - way ANOVA . ( * * p ≤ 0 . 01 ) . Values have first been normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . ( D ) . RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase ( control ) or against Kap1 ( Kap1 KD ) . Left : Cells with no ( 0 , red ) , one ( 1 , grey ) and two or more ( 2 , black ) Tsix foci were counted in two independent experiments , shown averaged . Statistical significance was determined by χ2 . A minimum of 110 cells were counted per each time point for each line ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Right : examples of cells with one ( top ) or two ( central and bottom ) Tsix FISH signals . Scale bars : 5μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. ((C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.(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.


Figure 5 ( A ) . ChIP - qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs ( Rif1 + / + , same as used in Fig . 3B but without OHT ) and during early differentiation . ZFP629 is a well - characterised KAP1 associated region ( positive control ) . Xite A and C indicate two regions within the Tsix enhancer Xite , Tsix region 1 indicates Tsix major promoter , Tsix region 2 indicates the Dxpas34 region , Tsix region 3 indicates a region slightly downstream of the Dxpas34 region . P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 is an intergenic region . See Appendix Fig . S2C for the positions of the primers within Xist and Tsix . The data are presented as mean ± standard deviation from three ( 2d EB and 1d EB ) and two ( ESCs ) independent experiments . Statistical significance was calculated by Student ' s two - tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB ( * p ≤ 0 . 05 and ns = not significant ) . In the inset , Tsix RNA levels were quantified by RT - qPCR during the differentiation of wild type female ESCs shown in Fig . 4F . The average of two experiments is shown . Tsix values are normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . Error bars indicate standard deviations . ( C ) . KAP1 association with Xist promoter in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female mESC cell lines . Ezr is an additional region known to be associated with KAP1 in mESCs . Enrichments are presented relative to input DNA . Mean ± standard deviation from a minimum of three independent experiments per cell line are displayed . Statistical significance was determined using Student ' s two - tailed , unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1 + / + versus Rif1 - / - cells ( * * p ≤ 0 . 01 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).


Figure 6 ( C ) . HEXIM1 association with the Xist promoter ( P1 and P2 ) in two independent Rif1 + / + and two Rif1 - / - mESC lines , analysed by ChIP - qPCR . Tkbp1 and Myf5 are two control regions . As in the case of KAP1 association , deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2 . Average ± standard deviation of two independent experiments . p values were calculated by two - tailed , unpaired , equal variance t - test . ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) . ( E ) . Upon infection of Fa2L cells with shRNA against Kap1 or control , against Luciferase , HEXIM1 association with P2 was analysed , on both alleles , by ChIP - qPCR . As in the case of KAP1 , HEXIM1 shows preferential association with Xist P2 on the Cast allele ( future Xa , Control ) . The association is lost upon knock down of Kap1 ( Kap1 KD ) . Myf5 serves as a negative and Neat1 as a positive control regions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C). HEXIM1 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and two Rif1-/- mESC lines, analysed by ChIP-qPCR. Tkbp1 and Myf5 are two control regions. As in the case of KAP1 association, deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2. Average ±standard deviation of two independent experiments. p values were calculated by two-tailed, unpaired, equal variance t-test. (**p ≤ 0.01, ***p ≤ 0.001).(E). Upon infection of Fa2L cells with shRNA against Kap1 or control, against Luciferase, HEXIM1 association with P2 was analysed, on both alleles, by ChIP-qPCR. As in the case of KAP1, HEXIM1 shows preferential association with Xist P2 on the Cast allele (future Xa, Control). The association is lost upon knock down of Kap1 (Kap1 KD). Myf5 serves as a negative and Neat1 as a positive control regions.