a , Survival ratio after TAC ( n = 7 - 14 per group ) .

a, Survival ratio after TAC (n = 7-14 per group).


b - g , Ten days after TAC . b , Gross appearance of hearts . Scale bar , 2 mm .

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b-g, Ten days after TAC. b, Gross appearance of hearts. Scale bar, 2 mm.


b - g , Ten days after TAC . c , Echocardiography . Scale bars , 0 . 2 s and 5 mm . Echocardiographic parameters ( d ) ( n = 7 - 13 per group ) . LVIDd and LVIDs , end - diastolic and end - systolic left ventricular internal dimension , respectively ; LVFS , left venticular fractional shortening ;

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b-g, Ten days after TAC. c, Echocardiography. Scale bars, 0.2 s and 5 mm. Echocardiographic parameters (d) (n = 7-13 per group). LVIDd and LVIDs, end-diastolic and end-systolic left ventricular internal dimension, respectively; LVFS, left venticular fractional shortening;


b - g , ( e ) Ten days after TAC . physiological parameters ( n = 7 - 13 per group ) .

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b-g, (e) Ten days after TAC.physiological parameters (n = 7-13 per group).


Haematoxylin and eosin - stained ( f ) and azocarmine and aniline blue ( AZAN ) - Mallory - stained ( g ) heart sections . Scale bar , 100 µm . Data are mean ± s . e . m . * P 0 . 05 versus all other groups , * * P 0 . 05 versus sham - operated controls .

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Haematoxylin and eosin-stained (f) and azocarmine and aniline blue (AZAN)-Mallory-stained (g) heart sections. Scale bar, 100 µm. Data are mean ± s.e.m. *P  0.05 versus all other groups, **P  0.05 versus sham-operated controls.


Mice were analysed 2 days after TAC ( a - c ) . a , Haematoxylin and eosin - stained heart sections . Scale bar , 100 µm . b , AZAN - Mallory - stained sections . Scale bar , 100 µm .

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Mice were analysed 2 days after TAC (a-c). a, Haematoxylin and eosin-stained heart sections. Scale bar, 100 µm. b, AZAN-Mallory-stained sections. Scale bar, 100 µm.


Mice were analysed 2 days after TAC ( a - c ) . c , Immunohistochemical analysis using antibodies to CD45 , CD68 , Ly6G and CD3 . Scale bar , 100 µm .

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Mice were analysed 2 days after TAC (a-c). c, Immunohistochemical analysis using antibodies to CD45, CD68, Ly6G and CD3. Scale bar, 100 µm.


Mice were analysed 2 days after TAC ( a - e ) . a , Electron microscopic analysis . Images of mitochondria at higher magnification are shown in subsets . Scale bar , 1 µm . b , Autolysosome after incubation with anti - DNA antibody and 10 nm gold staining . Scale bar , 200 nm . Arrows indicate labelled DNA .

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Mice were analysed 2 days after TAC (a-e). a, Electron microscopic analysis. Images of mitochondria at higher magnification are shown in subsets. Scale bar, 1 µm. b, Autolysosome after incubation with anti-DNA antibody and 10 nm gold staining. Scale bar, 200 nm. Arrows indicate labelled DNA.


Mice were analysed 2 days after TAC Double staining of heart sections with EdU ( green ) and anti - LAMP2a antibody ( red ) ( c ) , EdU ( green ) and anti - LC3 antibody ( red ) ( d ) or EdU and anti - TLR9 antibody ( red ) ( e ) . Arrows indicate EdU - positive and LAMP2a - , LC3 - or TLR9 - positive structures . Scale bar , 10 µm .

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Mice were analysed 2 days after TAC Double staining of heart sections with EdU (green) and anti-LAMP2a antibody (red) (c), EdU (green) and anti-LC3 antibody (red) (d) or EdU and anti-TLR9 antibody (red) (e). Arrows indicate EdU-positive and LAMP2a-, LC3- or TLR9-positive structures. Scale bar, 10 µm.


a , Survival ratio of TAC - operated ODN - treated mice ( n = 6 - 10 per group ) .

a, Survival ratio of TAC-operated ODN-treated mice (n = 6-10 per group).


b - d , Four days after TAC . b , Echocardiography . Scale bars , 0 . 2 s and 5 mm .

b-d, Four days after TAC. b, Echocardiography. Scale bars, 0.2 s and 5 mm.


b - d , Four days after TAC . c , Echocardiographic parameters . Open and closed bars represent ODN2088 control - and ODN2088 - treated groups , respectively ( n = 5 - 8 per group ) .

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b-d, Four days after TAC. c, Echocardiographic parameters. Open and closed bars represent ODN2088 control- and ODN2088-treated groups, respectively (n = 5-8 per group).


b - d , Four days after TAC . d , Immunohistochemical analysis . Scale bar , 100 µm .

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b-d, Four days after TAC. d, Immunohistochemical analysis. Scale bar, 100 µm.


TLR9 - deficient mice were analysed 10 weeks after TAC ( e , f ) . e , Scale bars , 0 . 2 s and 5 mm . f , Echocardiographic parameters ( n = 6 - 10 per group ) . Data are mean ± s . e . m . * P 0 . 05 versus all other groups .

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TLR9-deficient mice were analysed 10 weeks after TAC (e, f). e, Scale bars, 0.2 s and 5 mm. f, Echocardiographic parameters (n = 6-10 per group). Data are mean ± s.e.m. *P  0.05 versus all other groups.


Western blots for exercise - associated phosphorylated proteins in ( top ) contracted ( + ) or sham ( - ) rat muscle and ( bottom ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total AMPK and 14 - 3 - 3 were used as loading controls .

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Western blots for exercise-associated phosphorylated proteins in (top) contracted (+) or sham (-) rat muscle and (bottom) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total AMPK and 14-3-3 were used as loading controls.


Volcano plots of phosphopeptide median Log2 fold - change ( contraction / sham surgery ) plotted against the - Log10 p - value in rat contraction ( left ) or mouse running ( right ) . Colored dots represent significantly - regulated phosphopeptides ( p < 0 . 05 , n = 5 , moderated t - test ) . Gray dots represent phosphopeptides that were detected but not significantly altered in exercise .

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Volcano plots of phosphopeptide median Log2 fold-change (contraction/sham surgery) plotted against the -Log10 p-value in rat contraction (left) or mouse running (right). Colored dots represent significantly-regulated phosphopeptides (p < 0.05, n=5, moderated t-test). Gray dots represent phosphopeptides that were detected but not significantly altered in exercise.


Western blots for STIM1 phosphorylated at S257 or S521 in ( left ) contracted ( + ) or sham ( - ) rat muscle and ( right ) muscle from mice that underwent a treadmill running protocol ( + ) or remained at rest ( - ) . Total STIM1 and 14 - 3 - 3 were used as loading controls . 14 - 3 - 3 data for mouse running was re - used from Figure 1B .

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Western blots for STIM1 phosphorylated at S257 or S521 in (left) contracted (+) or sham (-) rat muscle and (right) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total STIM1 and 14-3-3 were used as loading controls. 14-3-3 data for mouse running was re-used from Figure 1B.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in L6 rat myoblasts with siRNA - mediated knockdown of endogenous STIM1 ( siSTIM1 ) or transfected with scramble control siRNA ( Control ) . Cells were treated with the AMPK activator A - 769662 ( + ) or vehicle ( - ) for 30 min . Total STIM1 was used to demonstrate STIM1 knockdown efficiency . Total ACC and 14 - 3 - 3 were used as loading controls .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK β1 / β2 knockout ( AMPKβ KO ) mouse endothelial fibroblasts were isolated and treated with A - 769662 ( + ) or vehicle ( - ) for 30 min . AMPKβ was used to demonstrate knockout , and AMPKɑ was used to demonstrate specificity for AMPKβ . Total ACC and STIM1 were used as loading controls .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.


Western blots for total ACC and STIM1 and phosphorylated STIM1 ( S257 and S521 ) and ACC ( S79 ) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP - STIM1 .

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Western blots for total ACC and STIM1 and phosphorylated STIM1 (S257 and S521) and ACC (S79) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP-STIM1.


Representative Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 and S521 ) in response to a 15 min treatment with increasing doses of thapsigargin ( . 01 , . 1 , 1 or 10 µM ) or . 5 mM AICAR . DMSO was used as a vehicle control . 14 - 3 - 3 was used as a loading control . n = 3 independent experiments .

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Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.


Western blots for phosphorylated ACC ( S79 ) and STIM1 ( S257 or S521 ) in wild type ( WT ) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin ( + ) or vehicle ( - ) for 15 min .

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Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . The mClover2 : tagRFP - T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. The mClover2:tagRFP-T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . STIM1 OASF expressed in live L6 myoblasts before ( - ) or after ( + ) treatment with A - 769662 .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.


Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2 . L6 myoblasts with S257 mutated to A or E .

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Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. L6 myoblasts with S257 mutated to A or E.


L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts ,

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L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts,


L6 myoblasts were transfected with the genetic cytosolic Ca2 + sensor GCaMP6s . Cells were also transfected with non - targeted siRNA ( Control ) or siRNA directed at endogenous STIM1 ( siSTIM1 ) . Some cells with endogenous STIM1 knockdown were also transfected with full - length wild type ( WT ) or mutant ( S257A , S257E or L251S ) STIM1 - mRuby3 . Ca2 + levels over time ( fold of 0 mM Ca2 + + EGTA basal ) and ( C ) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2 + and treated with 2 µM thapsigargin during the indicated times .

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L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.


Negative geotaxis climbing assays in Drosophila ( flies ) . Time required for 50 % of flies to climb to the target line before or after 2 . 5 hr of continuous climbing exercise ( A ) in the yw strain with muscle - specific STIM knockdown using two independent RNAi sequences ( RNAi26 - 1 or RNAi8 - 4 ) compared to GFP - expressing controls , or ( B ) in the w1118 strain with muscle - specific STIM knockdown using RNAiGD16187 compared to non - targeted RNAiGD12145 controls .

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Negative geotaxis climbing assays in Drosophila (flies). Time required for 50% of flies to climb to the target line before or after 2.5 hr of continuous climbing exercise (A) in the yw strain with muscle-specific STIM knockdown using two independent RNAi sequences (RNAi26-1 or RNAi8-4) compared to GFP-expressing controls, or (B) in the w1118 strain with muscle-specific STIM knockdown using RNAiGD16187 compared to non-targeted RNAiGD12145 controls.


Time to fatigue , when flies were no longer able to climb , during a 3 - min period after negative geotaxis stimulation in STIM whole - body knockout flies overexpressing wild type ( WT ) or mutant ( S257A or S257E ) human STIM1 - mRuby3 .

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Time to fatigue, when flies were no longer able to climb, during a 3-min period after negative geotaxis stimulation in STIM whole-body knockout flies overexpressing wild type (WT) or mutant (S257A or S257E) human STIM1-mRuby3.


( A ) Coimmunoprecipitation of SMO - HA and Myc - Smaug proteins . Extracts of Cl8 cells expressing combinations of SMOWT - HA or SMOPKA - SD - HA with Myc - SmaugWT in the absence or presence of HH were immunoprecipitated ( IP ) with anti - HA . The input ( lower panel ) and the IP complexes ( upper panel ) were analyzed by western blot with anti - Myc ( αMyc ) or anti - HA ( αHA ) antibodies . Here and in the other figures , the names of the proteins detected are indicated on the left and the molecular weights on the right , in kDa ; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs . * corresponds to higher molecular weight forms of SMO of unknown origin . Here and in panel F , the black arrow indicates unphosphorylated SMOWT - HA and the bracket indicates the phosphorylated forms of SMOWT - HA or SMOPKA - SD .

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(A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD.


( D , E ) Mapping of the Smaug interaction domain in SMO . Extracts from Cl8 cells transfected with Myc - SmaugWT and various regions of SMO fused to either HA ( D ) or to GFP ( E ) were immunoprecipitated with αMyc ( D ) or αGFP ( E ) and analyzed by western blotting with αMyc ( lower panel in D and upper panel in E ) , αHA ( D , upper panel ) or αGFP ( E , lower panel ) . In : Input ; Sup : supernatant . See also Fig EV1D - F .

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(D, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F.


( F ) Hyperphosphorylated forms of SMO do not interact with Smaug . Left panel : Extracts of Cl8 cells expressing SMOWT - HA with or without Myc - SmaugWT , in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA ( upper panel ) or αMyc antibodies ( lower panel ) . Right panel : Cl8 cells expressing SNAP - SMOWT with Myc - SmaugWT in the presence of HH were extracellularly labelled with a membrane - nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation ( IP ) with anti - Myc . Labelled SNAP - SMO was directly visualized after electrophoresis . Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms . See also Fig EV1G - J .

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(F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J.


( G ) SMO and Smaug colocalize . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT alone ( 1 - 2 ) or together with SMOWT - mCherry ( 3 - 3 ' ' and 4 - 4 ' ' ) either without ( 1 , 3 - 3 " ) or with HH ( 2 , 4 - 4 " ) . The merge images in 3 " and 4 " show GFP - SmaugWT in green and SMOWT - mCh in red . The same results were seen with different fluorescent tags as well ( Fig EV2B ) . The lack of effect of HH on GFP - SmaugWT in absence of SMOWT - mCh is likely due to limiting amounts of endogeneous SMO . A least 20 cells were assayed for each condition . In absence of HH , all co - transfected cells exhibited greater than 90 % colabelled SMOWT - mCherry . Scale bar ( shown in G1 , identical for all panels ) : 10μm . See also Fig EV2 .

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(G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.


( B , C ) Smaug downregulates reporter expression . Mean values of the relative levels of SNAP - GUS reporter ( SNAP - GUS / GFP - SNAP ratio , in arbitrary units ) in extracts of transfected Cl8 cells in the absence ( black ) or presence of λN - SNAP - Smaug ( red ) , λN - SNAP ( pale grey ) or SNAP - Smaug ( dark grey ) . See Fig EV3A for the representative gel and Fig EV3B which shows that λN - SNAP - Smaug has no effect on a SNAP - GUS construct that lacks the 5BoxB . N = 3 ( biological replicates ) . See also Fig EV3A - C . Here and in D - F : SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug . The error bars correspond to the standard deviation of the mean ( SD ) . Statistical analysis was done by a Kruskal - Wallis test by ranks followed by a Dunn test and p values are : black bracket < 0 . 05 , red bracket < 0 . 01 , green bracket < 0 . 0001 . Here and in D - E : 15 ng of pAct . λN - SNAP - smaug plasmid were used per transfection .

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(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection.


, E ) HH / SMO reduces Smaug levels and its overall negative effect on reporter expression . Mean values of the relative levels of reporter expression ( determined as above ) ( D ) or of λN - SNAP - Smaug ( ratio λN - SNAP - Smaug / GFP - SNAP ) ( E ) in the absence of SMO constructs ( red ) or in the presence of SMOWT - HA ( green ) , SMOPKA - SD - HA ( blue ) or SMO ∆ 958 - HA ( yellow ) , without HH ( plain boxes ) or in presence of HH ( striped boxes ) . N = 6 ( independent biological replicates ) , except for " no SMO , no HH " and " SMOWT with HH " conditions where n = 10 ( independent biological replicates ) . A representative gel is shown in Fig EV3D . Note that SMOWT - HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant . See also Fig EV3E - F .

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, E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.


( F ) HH / SMO reduces Smaug ' s intrinsic repressive ability . A similar experiment was conducted with different amounts of λN - SNAP - smaug expressing plasmid ( ranging from 0 to 50 ng ) as indicated ) . The mean values of the relative levels of reporter ( y - axis ) are plotted against the relative levels of λN - SNAP - Smaug ( x - axis ) . SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug in absence of SMOPKA - SD / HH . The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with ( blue ) and without ( red ) SMOPKA - SD / HH , respectively . N = 3 ( biological triplicates ) . See Fig EV3H and also a representative gel in Fig EV3G .

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(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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

(A 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 .

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(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 .

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

(A 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 ) :

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(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


( 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

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(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 .

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(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 .

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(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.


C ) 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 )

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C) 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)


A ) 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 .

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

A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) 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


A , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,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.


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

B) 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.


C ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) 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


B ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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


A ) 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 )

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

A) 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A ) Mitochondrial pH at CV SU e is compared for HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions .

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

A) 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 .

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

D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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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 .

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


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

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


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

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


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

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


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

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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 .

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


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

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(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 .

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


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

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(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 .

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


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

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(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 ) .

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


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

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(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 ) .

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


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

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(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 ) .

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(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 ) .

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


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

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


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

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


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

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


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

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


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

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


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

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