F ) Quantification of long - range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two - minute movie ( n = 18 cells per condition over three independent experiments ) . Data information : Statistical significance in F ) was calculated by two - way ANOVA . For F ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments). Data information: Statistical significance in F) was calculated by two-way ANOVA. For F) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


G ) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells .

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G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.


H ) Quantification of long - range peroxisomal trafficking events ( visualised with pxGFP ) in HeLa cells transfected with scrambled , Miro1 and Pex14 siRNA , by blind scoring of a five - minute movie ( n = 18 cells over three independent experiments ) . Data information : For H ) is a one - way ANOVA with Newman - Keuls post - hoc test . * * denotes p < 0 . 01 . For H ) , no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments). Data information: For H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


A ) Representative images of WT , Miro1KO , Miro2KO and DKO MEFs seeded onto Y - shaped fibronectin micropatterns stained for mitochondria ( Tom20 in red ) and peroxisomes ( Catalase in white ) . Scale bar represents 10 μm . Data information : n = 60 cells over three independent experiments .

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A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm. Data information: n = 60 cells over three independent experiments.


B ) Normalised cumulative distribution of mitochondria in WT , Miro1KO , Miro2KO and DKO MEFs . C ) Distance at which 95 % of mitochondria are distributed ( Mito95 ) for all four genotypes of MEF . Data information : n = 60 cells over three independent experiments . For C ) one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

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B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs. C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF. Data information: n = 60 cells over three independent experiments. For C) one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


D ) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT , Miro1KO , Miro2KO and DKO MEFs . E ) Bar graph comparing the average distance at which 95 % of the peroxisomal signal is distributed ( Perox95 ) . Data information : n = 60 cells over three independent experiments . For E ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

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D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs. E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95). Data information: n = 60 cells over three independent experiments. For E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


A ) Snapshots and tracks of pxDsRed signal in WT , Miro1KO , Miro2KO and DKO MEFs following live imaging at 1 . 5 seconds per frame for two minutes . B ) Quantification of median net displacement of individual peroxisomes ( pxDsRed signal ) for WT , Miro1KO , Miro2KO and DKO MEFs ( n = 42 cells over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For multiple comparisons in B ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance .

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A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.


C ) Still images every 30 seconds of ER and peroxisomes ( ER - DsRed pseudo - coloured to green and pxGFP pseudo - coloured to magenta , respectively ) in WT and DKO MEFs taken from two - minute movies by live - cell spinning - disk microscopy . Arrows track individual peroxisomes associated with an ER tubule . Scale bar is 5 μm .

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C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.


D ) Quantification of the relative ER displacement over a two - minute movie using ER - DsRed signal in WT and DKO MEFs ( n = 20 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


E ) Median net displacement of peroxisomes in WT and DKO MEFs . n = 20 cells per condition over three independent experiments .

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E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.


F ) Median net displacement of peroxisomes in DKO MEFs with and without myc - tagged variant 4 of Miro1 ( mycv4 ) ( n = 12 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


A ) Representative images of catalase staining ( peroxisomes ) in WT , Miro1KO , Miro2KO and DKO MEFs .

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A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.


B ) Bar graph comparing average peroxisomal area of WT Miro1KO , Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in B ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


C ) Comparison of total peroxisomal number between WT , Miro1KO , Miro2KO and DKO MEFs . n = 60 cells per condition over three independent experiments . Data information : Calculation of statistical significance in C ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


D ) Representative images of Control ( GFP - tagged 1 - 70 of Tom70 ) , GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase ( peroxisomes ) .

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D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).


E ) Representative images of Pex14 ( peroxisomes ) in DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) .

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E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).


F ) Bar graph comparing average peroxisomal area of Control , GFPMiro1 and GFPMiro2 overexpressing WT MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in F ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


G ) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) ( n = 30 cells per condition over three independent experiments ) . Data information : Statistical test used in G ) was a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


A ) Representative images of proximity - ligation assay ( PLA ) of Fis1 and Drp1 in WT and DKO MEFs . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . B ) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs . Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls ( n = 3 experiments ) . Data information : For B ) statistical significance was quantified by a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


C ) Representative images of a Fis1 - Drp1 PLA in DKO MEFs expressing either GFP , GFPv1 ( GFP - tagged variant 1 of Miro1 ) or GFPv4 ( GFP - tagged variant 4 of Miro1 ) n = 30 cells per condition over three independent experiments ) . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . D ) Quantification of PLA fluorescent dots per cells between GFP , GFPv1 or GFPv4 transfected DKO MEFs ( n = 30 cells over three independent experiments ) . Data information : In D ) a one - way ANOVA with a Newman - Keuls post - hoc test was used to calculate statistical significance . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


E ) Western blot of Drp1 , Fis1 and actin from WT and DKO whole cell lysates . F ) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs ( n = 3 ) . Data information F ) , statistical significance was quantified by a two - tailed Student ' s t test .

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E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates. F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3). Data information F), statistical significance was quantified by a two-tailed Student's t test.


G ) Zooms of endogenous Drp1 , mitochondria ( Mitotracker ) and peroxisomes ( PMP70 ) in WT and DKO MEFs . Scale bar is 5 μm . H ) Integrated density of Drp1 signal on Mitotracker - positive and PMP70 - negative structures in WT and DKO MEFs . I ) Integrated density of Drp1 signal on PMP70 - positive and Mitotracker - negative structures in WT and DKO MEFs . Data information G - I n = 42 cells per condition over three independent experiments . For , H ) statistical significance was quantified by a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm. H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs. I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs. Data information G-I n=42 cells per condition over three independent experiments. For , H) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


( B - C ) Representative confocal images and their quantification of Gb3 accumulation within the lysosome , detected by fluorescent - conjugated Shiga toxin , from WT , CLN3 - KO and CLN7 - KO ARPE - 19 cells treated with PDMP or silenced for Gb3 synthase ( siGb3S ) ( * * * vs WT , ° ° ° vs DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(B-C) Representative confocal images and their quantification of Gb3 accumulation within the lysosome, detected by fluorescent-conjugated Shiga toxin, from WT, CLN3-KO and CLN7-KO ARPE-19 cells treated with PDMP or silenced for Gb3 synthase (siGb3S) (*** vs WT, °°° vs DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( A - B ) Representative confocal images of Gb3 accumulation , revealed by STX staining , in brain sections from CLN7Δex2 mice at 3 and 7 . 5 months of age compared with CLN7 WT mice . Scale bars : 80 µm .

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(A-B) Representative confocal images of Gb3 accumulation, revealed by STX staining, in brain sections from CLN7Δex2 mice at 3 and 7.5 months of age compared with CLN7 WT mice. Scale bars: 80 µm.


( E ) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain . Data are presented as mean ± SD , * * : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas )

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(E) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain. Data are presented as mean ± SD, **: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (n= 3 biological replicas)


( A ) Representative confocal images and quantification of SCMAS staining within the lysosome of ARPE - 19 WT and CLN3 KO upon depletion of Gb3S ( siGb3S ) , LacCer ( siLCS ) , and GM3s ( siGM3S ) . ( * * * vs WT , ° ° ° vs CLN3 KO siSCR ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(A) Representative confocal images and quantification of SCMAS staining within the lysosome of ARPE-19 WT and CLN3 KO upon depletion of Gb3S (siGb3S), LacCer (siLCS), and GM3s (siGM3S). (*** vs WT, °°° vs CLN3 KO siSCR). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( D ) Representative confocal images and quantification of STX staining within the lysosome of ARPE - 19 WT and CLN7 KO in DMSO or treated 48h with Tamoxifen . ( * * * vs WT , ° ° ° vs CLN3 KO DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(D) Representative confocal images and quantification of STX staining within the lysosome of ARPE-19 WT and CLN7 KO in DMSO or treated 48h with Tamoxifen. (*** vs WT, °°° vs CLN3 KO DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( E ) Representative confocal images and quantification of STX staining within the lysosome of NPCs WT and derived from CLN7Pa474 patient IPSCs in DMSO or treated with Tamoxifen for 48h . ( * * * vs WT , ° ° ° vs CLN7Pa474 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(E) Representative confocal images and quantification of STX staining within the lysosome of NPCs WT and derived from CLN7Pa474 patient IPSCs in DMSO or treated with Tamoxifen for 48h. (*** vs WT, °°° vs CLN7Pa474 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( A ) Representative confocal images and Quantification of STX within the lysosome in U2 - OS and HeLa cells after acute silencing of CLN3 ( siCLN3 ) in DMSO or treated 48h with Tamoxifen . ( * * * vs siSCR DMSO , ° ° ° vs siCLN3 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal images and Quantification of STX within the lysosome in U2-OS and HeLa cells after acute silencing of CLN3 (siCLN3) in DMSO or treated 48h with Tamoxifen. (*** vs siSCR DMSO, °°° vs siCLN3 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( B ) Representative confocal image and quantification of Gb3 in ARPE - 19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB ( siTFEB ) for 72h and treated for the last 48h with DMSO or Tamoxifen . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(B) Representative confocal image and quantification of Gb3 in ARPE-19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB (siTFEB) for 72h and treated for the last 48h with DMSO or Tamoxifen. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( C ) Representative confocal image and quantification of TFEB in U2 - OS cells silenced with siRNA against scramble sequence and CLN3 ( siCLN3 ) for 72h and treated for the last 48h with DMSO or Tamoxifen ( 5 µM and 10 µM ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(C) Representative confocal image and quantification of TFEB in U2-OS cells silenced with siRNA against scramble sequence and CLN3 (siCLN3) for 72h and treated for the last 48h with DMSO or Tamoxifen (5 µM and 10 µM). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( C ) Representative confocal image and quantification of TFEB localization in HeLa TFEB - GFP transfected with RagC for 48h and treated for the last 3h with DMSO , Torin1 , Tamoxifen or Ospemifene . Ratios of nuclear to cytosolic TFEB localization in RagC non - expressing ( RagC - ) and RagC - expressing cells ( RagC + ) are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(C) Representative confocal image and quantification of TFEB localization in HeLa TFEB-GFP transfected with RagC for 48h and treated for the last 3h with DMSO, Torin1, Tamoxifen or Ospemifene. Ratios of nuclear to cytosolic TFEB localization in RagC non-expressing (RagC-) and RagC-expressing cells (RagC+) are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


D ) Representative confocal images and quantification of STX in ARPE - 19 CLN3 KO cells transfected with empty vector or HA - RagC for 48h and treated for 48h with DMSO or Tamoxifen . STX average spot area presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

D) Representative confocal images and quantification of STX in ARPE-19 CLN3 KO cells transfected with empty vector or HA-RagC for 48h and treated for 48h with DMSO or Tamoxifen. STX average spot area presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Representative confocal images of STX in the Cortex , Hippocampus , and Cerebellum brain section derived from 7 . 5 months - old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen ( Tamox ) . Quantification of confocal images , the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° / ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * / oo : P ≤ 0 . 001 , * * * / ooo : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 60 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Representative confocal images of STX in the Cortex, Hippocampus, and Cerebellum brain section derived from 7.5 months-old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen (Tamox). Quantification of confocal images, the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°°/°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, **/oo: P ≤ 0.001, ***/ooo: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 60 µm.


Representative confocal images of SCMAS in the Cortex , Hippocampus , and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen . Quantification of confocal images , the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological ) . Scale bars : 60 µm .

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

Representative confocal images of SCMAS in the Cortex, Hippocampus, and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen. Quantification of confocal images, the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological). Scale bars: 60 µm.


( A - B ) Representative confocal images and quantification of IBA - 1 in the Cortex , Hippocampus , and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen . ( * * * / * * / * vs WT , ° ° ° / ° ° / ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * / o : P ≤ 0 . 01 , * * / oo : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 50 µm .

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

(A-B) Representative confocal images and quantification of IBA-1 in the Cortex, Hippocampus, and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm.


( D ) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen .

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

(D) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . A Representative H & E staining from antral glands ( upper row ) . The dotted line marks the gland base cell area , which expands upon infection in Lgr5 - / - and control mice . Immunofluorescence labeling for GSII and Ki67 ( middle row ) and GIF and Muc5ac ( lower row ) of antral epithelium from Lgr5 - / - and control mice uninfected and after 2 months of infection . Scale bars : 50 µm .

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

Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which expands upon infection in Lgr5-/- and control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (lower row) of antral epithelium from Lgr5-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . B Quantification of antral gland height in Lgr5 - / - compared to control mice . Uninfected control ( n = 5 ) versus uninfected Lgr5 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr5 - / - ( n = 4 ) . Data information : B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for B ,

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

Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr5-/- compared to control mice. Uninfected control (n = 5) versus uninfected Lgr5-/- (n = 4) versus infected control (n = 4) versus infected Lgr5-/- (n = 4). Data information: B, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for B,


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . C Quantifications of GSII + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) , Ki67 as percentage of total DAPI - stained cells ( n = 3 - 4 biological replicates per group , n = 20 glands per mouse ) , GIF + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) , Muc5ac + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) . Data information : For ( one - way ANOVA + Tukey ´ s multiple comparison test for C ,

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

Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 3-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For (one-way ANOVA + Tukey´s multiple comparison test for C,


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . D H . pylori CFUs in Lgr5 - / - ( n = 5 ) and control mice ( n = 5 ) . Data information : For D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - test for

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

Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs in Lgr5-/- (n = 5) and control mice (n = 5). Data information: For D, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for D


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . E qPCR expression levels of Lgr4 , Lgr5 , Axin2 , gland base cell markers ( Gif , Pgc , Tff2 , Muc6 ) , pit cell marker Muc5ac , and Itln1 in uninfected Lgr5 - / - ( n = 6 - 10 ) and control mice ( n = 4 - 10 ) . F qPCR expression levels of Lgr4 , Lgr5 , gland base cell markers ( Gif , Pgc , Muc6 ) , and Itln1 in Lgr5 - / - ( n = 5 ) and control mice ( n = 5 ) after 2 months of infection . Data information : For E and F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - test for E and F ) .

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

Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr5-/- (n = 6-10) and control mice (n = 4-10). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr5-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for E and F).


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . A Representative H & E staining from antral glands ( upper row ) . The dotted line marks the gland base cell area , which does not expand in infected Lgr4 - / - but does expand in the corresponding control mice . Immunofluorescence labeling for GSII and Ki67 ( middle row ) and GIF and Muc5ac ( bottom row ) of antral epithelium from Lgr4 - / - and control mice uninfected and after 2 months of infection . Scale bars : 50 µm .

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

Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which does not expand in infected Lgr4-/- but does expand in the corresponding control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (bottom row) of antral epithelium from Lgr4-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . B Quantification of antral gland height in Lgr4 - / - compared to control mice . Uninfected control ( n = 2 ) versus uninfected Lgr4 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr4 - / - ( n = 4 ) . Data information : For B , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for

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

Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr4-/- compared to control mice. Uninfected control (n = 2) versus uninfected Lgr4-/- (n = 4) versus infected control (n = 4) versus infected Lgr4-/- (n = 4). Data information: For B, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for B


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . C Quantifications of GSII + area ( n = 2 - 4 biological replicates per group , n = 10 glands per mouse ) , Ki67 as percentage of total DAPI - stained cells ( n = 2 - 4 biological replicates per group , n = 20 glands per mouse ) , GIF + area ( n = 2 - 4 biological replicates per group , n = 10 glands per mouse ) , Muc5ac + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for C ,

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

Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 2-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for C,


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . D H . pylori CFUs show higher colonization in Lgr4 - / - ( n = 5 ) compared to control mice ( n = 4 ) . Data information : For D , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - test for

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

Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs show higher colonization in Lgr4-/- (n = 5) compared to control mice (n = 4). Data information: For D, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for D


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . E qPCR expression levels of Lgr4 , Lgr5 , Axin2 , gland base cell markers ( Gif , Pgc , Tff2 , Muc6 ) , pit cell marker Muc5ac , and Itln1 in uninfected Lgr4 - / - ( n = 4 - 10 ) and control mice ( n = 4 - 6 ) . F qPCR expression levels of Lgr4 , Lgr5 , gland base cell markers ( Gif , Pgc , Muc6 ) , and Itln1 in Lgr4 - / - ( n = 5 ) and control mice ( n = 5 ) after 2 months of infection . Data information : For E and F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - test for E and F ) .

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

Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr4-/- (n = 4-10) and control mice (n = 4-6). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr4-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for E and F).


A , B GSEA comparing microarray data from 2 - month infected Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) mice with publicly available data demonstrating downregulation of antral stem cell signature ( A ) and beta - catenin targets ( B ) .

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A, B GSEA comparing microarray data from 2-month infected Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) mice with publicly available data demonstrating downregulation of antral stem cell signature (A) and beta-catenin targets (B).


C qPCR expression levels for beta catenin targets Axin2 and Sox9 in uninfected and infected Lgr4 - / - and control mice ( n = 2 - 9 ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) .

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

C qPCR expression levels for beta catenin targets Axin2 and Sox9 in uninfected and infected Lgr4-/- and control mice (n = 2-9). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


D GSEA comparing microarray data from 2 - month infected Lgr4 - / - mice with publicly available data demonstrating downregulation of the TNF - α signaling via NF - κB .

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D GSEA comparing microarray data from 2-month infected Lgr4-/- mice with publicly available data demonstrating downregulation of the TNF-α signaling via NF-κB.


E Immunofluorescence labeling for p65 , Ki67 and GSII in the mouse antrum tissue from uninfected and infected Lgr4 - / - and control mice . Insets show magnified images . Scale bars : 50 µm .

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E Immunofluorescence labeling for p65, Ki67 and GSII in the mouse antrum tissue from uninfected and infected Lgr4-/- and control mice. Insets show magnified images. Scale bars: 50 µm.


F Quantification of p65 + area as percentage of total DAPI - stained cells of uninfected and infected Lgr4 - / - and control mice ( n = 4 biological replicates per group , n = 20 glands per mouse ) . Data information : For data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) .

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

F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Lgr4-/- and control mice (n = 4 biological replicates per group, n = 20 glands per mouse). Data information: For data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


G Immunofluorescence co - localization of p65 acetyl K310 ( activated form of NF - κB ) in the mouse antrum tissue of infected Lgr4 - / - and in control mice . Insets show magnified images . Scale bars : 50µm . Data information : For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates .

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G Immunofluorescence co-localization of p65 acetyl K310 (activated form of NF-κB) in the mouse antrum tissue of infected Lgr4-/- and in control mice. Insets show magnified images. Scale bars: 50µm. Data information: For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.


H qPCR expression levels of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in uninfected and infected Lgr4 - / - mice ( n = 4 - 5 ) and control mice ( n = 2 - 5 ) . Data information : For H , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) .

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

H qPCR expression levels of Cxcl1, Cxcl2, Cxcl10, Ccl2 in uninfected and infected Lgr4-/- mice (n = 4-5) and control mice (n = 2-5). Data information: For H, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


I Immunofluorescence labeling for Cxcl1 in the mouse antrum tissue in infected Lgr4 - / - and in control mice . Scale bars : 50 µm . Data information : For I images represent findings that were reproduced at least twice in the laboratory in independent biological replicates .

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I Immunofluorescence labeling for Cxcl1 in the mouse antrum tissue in infected Lgr4-/- and in control mice. Scale bars: 50 µm. Data information: For I images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.


A Immunofluorescence labeling for p65 and E - cadherin in the mouse antrum tissue of 2 weeks infected B6 - Myh11CreErt2 / Rspondin3fl / fl ( Rspo3 - / - ) and control mice . Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3 - / - mice . Scale bars : 50 µm .

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A Immunofluorescence labeling for p65 and E-cadherin in the mouse antrum tissue of 2 weeks infected B6-Myh11CreErt2/Rspondin3fl/fl (Rspo3-/-) and control mice. Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3-/- mice. Scale bars: 50 µm.


B Quantification of p65 + area as percentage of total DAPI - stained cells of infected Rspo3 - / - and control mice ( n = 3 biological replicates per group , n = 20 glands per mouse ) . Data information : For B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test for

O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of p65+ area as percentage of total DAPI-stained cells of infected Rspo3-/- and control mice (n = 3 biological replicates per group, n = 20 glands per mouse). Data information: For B, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for B


C qPCR expression level of Cxcl1 and Cxcl2 in 2 - week infected Rspo3 - / - mice ( n = 3 ) versus infected control mice ( n = 4 ) . D qPCR expression level of Cxcl1 and Cxcl2 in 2 - month infected Rspo3 - / - mice ( n = 5 ) versus infected control mice ( n = 6 ) . Data information : For C , D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test for C and

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR expression level of Cxcl1 and Cxcl2 in 2-week infected Rspo3-/- mice (n = 3) versus infected control mice (n = 4). D qPCR expression level of Cxcl1 and Cxcl2 in 2-month infected Rspo3-/- mice (n = 5) versus infected control mice (n = 6). Data information: For C, D, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for C and D


E Myh11CreERT2 / Rosa26Sor6 ( CAG - Rspo3 ) ( Rspo3 KI ) mice were infected for 6 weeks with H . pylori . Immunofluorescence labeling for p65 , GSII and KI67 in uninfected and infected Rspo3 KI and control mice . Scale bars : 50 µm . F Quantification of p65 + area as percentage of total DAPI - stained cells of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . G Quantification of GSII + area of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . H Quantification of Ki67 as percentage of total DAPI - stained cells of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . Data information : For , F , G and H data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant one - way ANOVA + Tukey ´ s multiple comparison test for F , G and H ) .

O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Myh11CreERT2/Rosa26Sor6(CAG-Rspo3) (Rspo3 KI) mice were infected for 6 weeks with H. pylori. Immunofluorescence labeling for p65, GSII and KI67 in uninfected and infected Rspo3 KI and control mice. Scale bars: 50 µm. F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). G Quantification of GSII+ area of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). H Quantification of Ki67 as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). Data information: For , F, G and H data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant one-way ANOVA + Tukey´s multiple comparison test for F, G and H).


I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice . Insets show magnified images . Scale bars : 50 µm .

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I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice. Insets show magnified images. Scale bars: 50 µm.


A Immunofluorescence labelling for MPO in uninfected and 2 - month infected Lgr4 - / - and control mice ( top panel ) and 2 - month infected Lgr5 - / - and control mice ( bottom panel ) . Asterisks indicate nonspecific staining . Scale bars : 50 µm . B Quantification of MPO + cells in uninfected and 2 - month infected Lgr4 - / - and control mice ( n = 2 biological replicates per group , n = 5 - 10 FOVs per mouse ) ( left panel ) and 2 - month infected Lgr5 - / - and control mice ( n = 2 biological replicates per group , n = 6 - 8 FOVs per mouse ) ( right panel ) . Data information : For B data are mean + SD . * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test ) .

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

A Immunofluorescence labelling for MPO in uninfected and 2-month infected Lgr4-/- and control mice (top panel) and 2-month infected Lgr5-/- and control mice (bottom panel). Asterisks indicate nonspecific staining. Scale bars: 50 µm. B Quantification of MPO+ cells in uninfected and 2-month infected Lgr4-/- and control mice (n = 2 biological replicates per group, n = 5-10 FOVs per mouse) (left panel) and 2-month infected Lgr5-/- and control mice (n = 2 biological replicates per group, n = 6-8 FOVs per mouse) (right panel). Data information: For B data are mean + SD. ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test).


Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . A Bright - field images of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 200 µm .

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Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. A Bright-field images of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 200 µm.


Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 50 µm .

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Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 50 µm.


Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions ( n = 4 - 5 biological replicates ) . D qPCR expression level of Gif in untreated organoids in respective medium conditions ( n = 4 - 5 biological replicates ) . Data information : For C , D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ( one - way ANOVA + Tukey ´ s multiple comparison test for C and Genes without asterisks are not significantly altered .

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

Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions (n = 4-5 biological replicates). D qPCR expression level of Gif in untreated organoids in respective medium conditions (n = 4-5 biological replicates). Data information: For C, D, data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 (one-way ANOVA + Tukey´s multiple comparison test for C and Genes without asterisks are not significantly altered.


Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . E qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within the respective medium conditions ( n = 6 biological replicates per condition ) . F qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in organoids after treatment with TNF - α compared to untreated controls within the respective medium conditions ( n = 2 - 3 biological replicates per condition ) . Data information : For E and F data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ; t - test for E and F ) . Genes without asterisks are not significantly altered .

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

Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. E qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within the respective medium conditions (n = 6 biological replicates per condition). F qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with TNF-α compared to untreated controls within the respective medium conditions (n = 2-3 biological replicates per condition). Data information: For E and F data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 ; t-test for E and F). Genes without asterisks are not significantly altered.


Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose was added for 2 h to all organoid conditions prior to analysis . A Electrophoretic mobility shift assay ( EMSA ) and SDS - PAGE western blot from whole cell protein ( WCL ) lysates from gastric organoids untreated or treated with ADP heptose , and maintained either in FM in medium without Rspo , without Wnt and Rspo , or + BMP2 . Representative images from n = 3 biological replicates . B Quantification of EMSA based on n = 3 biological replicates using ImageJ with normalization to loading ( ɑ - Tubulin ) . Data information : For B data are mean + SD . Significance is calculated by one - way ANOVA , F = 9 . 21 .

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

Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose was added for 2 h to all organoid conditions prior to analysis. A Electrophoretic mobility shift assay (EMSA) and SDS-PAGE western blot from whole cell protein (WCL) lysates from gastric organoids untreated or treated with ADP heptose, and maintained either in FM in medium without Rspo, without Wnt and Rspo, or +BMP2. Representative images from n = 3 biological replicates. B Quantification of EMSA based on n = 3 biological replicates using ImageJ with normalization to loading (ɑ-Tubulin). Data information: For B data are mean + SD. Significance is calculated by one-way ANOVA, F = 9.21.


C EMSA supershift from FM + ADP heptose organoids with the antibodies indicated . Blue arrow indicates NF - κB binding . Red arrow points to gel expected locations of supershifts from antibodies against p65 , cRel , or RelB . Grey arrow points to supershift location on the gel from antibodies against p52 or p50 . IgG lane serves as a negative control .

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C EMSA supershift from FM + ADP heptose organoids with the antibodies indicated. Blue arrow indicates NF-κB binding. Red arrow points to gel expected locations of supershifts from antibodies against p65, cRel, or RelB. Grey arrow points to supershift location on the gel from antibodies against p52 or p50. IgG lane serves as a negative control.


A Bright - field images of antrum organoids from 129 ; 129P2 - ctnnb1tm ( NFKBIAΔN ) 1Rsu ( ΔN ) mice and control mice , passage 0 , day 6 . Scale bars : 500 µm .

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A Bright-field images of antrum organoids from 129;129P2-ctnnb1tm(NFKBIAΔN)1Rsu (ΔN) mice and control mice, passage 0, day 6. Scale bars: 500 µm.


B , C qPCR expression levels of Lgr4 , Lgr5 , Gif , Pgc , Muc6 ( B ) and Cxcl1 , Cxcl2 ( C ) of organoids ( n = 3 biological replicates ) . Data information : For B , C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test ) .

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B, C qPCR expression levels of Lgr4, Lgr5, Gif, Pgc, Muc6 (B) and Cxcl1, Cxcl2 (C) of organoids (n = 3 biological replicates). Data information: For B, C, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).


D Representative H & E staining and immunofluorescence labeling for GSII , Ki67 and MUC5AC in uninfected ΔN and control mice . Scale bars : 50 µm . E Quantification of GSII and Ki67 as percentage of total DAPI - stained cells in ΔN and control mice ( n = 5 biological replicates per group , n = 20 glands per mouse ) . Data information : For E data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test ) .

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D Representative H&E staining and immunofluorescence labeling for GSII, Ki67 and MUC5AC in uninfected ΔN and control mice. Scale bars: 50 µm. E Quantification of GSII and Ki67 as percentage of total DAPI-stained cells in ΔN and control mice (n = 5 biological replicates per group, n = 20 glands per mouse). Data information: For E data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).


B . Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ , NRF1 , MVH , or MitoPLD .

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B. Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ, NRF1, MVH, or MitoPLD.


C . Localization of GFP fused with wild - type or mutant MLS of GASZ in HeLa cells . Mitochondrial localization was monitored by MitoTracker ( Red ) staining . Scale bars : 15 m .

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C. Localization of GFP fused with wild-type or mutant MLS of GASZ in HeLa cells. Mitochondrial localization was monitored by MitoTracker (Red) staining. Scale bars: 15 m.


A . IHF of GASZ and Cytochrome C ( Cyt C ) of testical sections from mice at day 7 postnatal , or MILI and MVH of gonad sections from wild - type embryos or embryos with MLS deletion at 16 . 5 dpc . Scale bars : 5 m .

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A. IHF of GASZ and Cytochrome C (Cyt C) of testical sections from mice at day 7 postnatal, or MILI and MVH of gonad sections from wild-type embryos or embryos with MLS deletion at 16.5 dpc. Scale bars: 5 m.


B . Western blots of GASZ expression of testes at day 5 postnatal .

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B. Western blots of GASZ expression of testes at day 5 postnatal.


C . Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild - type littermates .

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C. Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild-type littermates.


D . Histological studies of testis sections from wild - type mice or mice with MLS deletion at different time points after birth . Scale bars : 50 m .

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D. Histological studies of testis sections from wild-type mice or mice with MLS deletion at different time points after birth. Scale bars: 50 m.


E . EM of testis sections from wild - type neonatal mice or mice with MLS mutation . Red arrows point to IMC among mitochondria .

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E. EM of testis sections from wild-type neonatal mice or mice with MLS mutation. Red arrows point to IMC among mitochondria.


F . Expression of transposable elements analyzed by real - time RT - PCR on testes from wild - type mice or mice with mutated MLS at various time points after birth . Data were shown as mean ( of expression from three biological replicates ) ± one s . e . m . n = 3 for each genotype . * * : p < 0 . 01 . * : p < 0 . 05 .

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F. Expression of transposable elements analyzed by real-time RT-PCR on testes from wild-type mice or mice with mutated MLS at various time points after birth. Data were shown as mean (of expression from three biological replicates) ± one s.e.m. n=3 for each genotype. **: p<0.01. *: p<0.05.


B . 293T cells with ectopically expressed proteins were extracted for soluble and membrane bound fraction and examined by Western blots .

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B. 293T cells with ectopically expressed proteins were extracted for soluble and membrane bound fraction and examined by Western blots.


C . Mitochondrial fraction from 293T cells ectopically expressing GASZ , MitoPLD , AIFM1 , or SIRT3 were treated in the presence or absence of Triton X - 100 and / or Proteinase K , and examined by Western blots .

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C. Mitochondrial fraction from 293T cells ectopically expressing GASZ, MitoPLD, AIFM1, or SIRT3 were treated in the presence or absence of Triton X-100 and/or Proteinase K, and examined by Western blots.


D . GST pull - down assay of GST - tagged GASZ with FLAG - GASZ produced from 293T cells .

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D. GST pull-down assay of GST-tagged GASZ with FLAG-GASZ produced from 293T cells.


E . Co - IP assays of FLAG or GFP - tagged GASZ in 293T cells . Antibodies for Co - IP ( IP ) or Western blots ( WB ) were listed on the left .

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E. Co-IP assays of FLAG or GFP-tagged GASZ in 293T cells. Antibodies for Co-IP (IP) or Western blots (WB) were listed on the left.


F . BiFC assays in which YFP was detected by flow cytometry in control 293T cells , or 293T cells co - expressed with GASZ fused with N - or C - terminal half of YFP . Ctrl : empty vector control .

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F. BiFC assays in which YFP was detected by flow cytometry in control 293T cells, or 293T cells co-expressed with GASZ fused with N- or C-terminal half of YFP. Ctrl: empty vector control.


G . Co - IP assays of GFP - GASZ with various FLAG - tagged wild - type GASZ or its deletion mutants in 293T

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G. Co-IP assays of GFP-GASZ with various FLAG-tagged wild-type GASZ or its deletion mutants in 293T cells


A . Mitochondria visualized by MitoTracker ( Red ) when wild - type GASZ or GASZ with MLS mutation was expressed in HeLa cells . Scale bar : 15 m .

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A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m.


B . EM on 293T cells with empty vector control ( Ctrl ) , wild - type GASZ , or GASZ with F to P mutation in MLS . Scale bar : 1 m . N : nucleus .

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B. EM on 293T cells with empty vector control (Ctrl), wild-type GASZ, or GASZ with F to P mutation in MLS. Scale bar: 1 m. N: nucleus.


C . Averaged percentage of HeLa cells ( determined from 100 cells in each experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with GASZ , its SAM / ZIP deletion mutant , or GASZ with MLS mutation .

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C. Averaged percentage of HeLa cells (determined from 100 cells in each experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with GASZ, its SAM/ZIP deletion mutant, or GASZ with MLS mutation.


D . HeLa cells transfected with COX4 - DsRed , mito - PAGFP , and empty vector control , GASZ or GASZ with MLS mutation , followed by photoactivation of PAGFP . Representative images of PAGFP and DsRed were taken at 0 , 10 , and 20 min post photoactivation . Scale Bar : 10 m . Insets show images with higher magnification . The averaged exponential decay constant ( ± s . e . m . ) of normalized PAGFP fluorescence within the designated region ( white circle ) at photoactivation area over a period of 10 min was shown as a bar graph below . n > 20 cells for each group .

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D. HeLa cells transfected with COX4-DsRed, mito-PAGFP, and empty vector control, GASZ or GASZ with MLS mutation, followed by photoactivation of PAGFP. Representative images of PAGFP and DsRed were taken at 0, 10, and 20 min post photoactivation. Scale Bar: 10 m. Insets show images with higher magnification. The averaged exponential decay constant (±s.e.m.) of normalized PAGFP fluorescence within the designated region (white circle) at photoactivation area over a period of 10 min was shown as a bar graph below. n>20 cells for each group.


E . Averaged percentage of C18 - 4 cells ( determined from 100 cells in experiment ) with different types of mitochondria determined by MitoTracker ( Red ) staining when transfected with Su9 - GFP or GFP - GASZ .

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E. Averaged percentage of C18-4 cells (determined from 100 cells in experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with Su9-GFP or GFP-GASZ.


F . Real - time PCR analyses of mtDNA copy numbers on testes at different time points postnatal , relative to a single copy gene , PECAM , encoded by nuclear DNA . n = 5 per genotype .

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F. Real-time PCR analyses of mtDNA copy numbers on testes at different time points postnatal, relative to a single copy gene, PECAM, encoded by nuclear DNA. n=5 per genotype.


G . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

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G. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


A - B . Co - IP assays of ectopically expressed FLAG - GASZ and MFN - HA in 293T cells ( A )

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A-B. Co-IP assays of ectopically expressed FLAG-GASZ and MFN-HA in 293T cells (A)


A - B . Co - IP assays of endogenous proteins of GASZ and MFNs from adult testes ( B ) .

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A-B. Co-IP assays of endogenous proteins of GASZ and MFNs from adult testes (B).


C . Co - IP assays of HA - tagged MFN1 with various FLAG - tagged wild - type GASZ , its domain deletion mutants , or GASZ with MLS mutations in 293T cells .

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C. Co-IP assays of HA-tagged MFN1 with various FLAG-tagged wild-type GASZ, its domain deletion mutants, or GASZ with MLS mutations in 293T cells.


D . Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker ( Red ) staining in wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid .

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D. Averaged percentage of MEFs with different types of mitochondria were determined according to the mitochondrial morphology by MitoTracker (Red) staining in wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid.


E . PEG fusion assays of Su9 - GFP and COX4 - DsRed labeled wild - type MEFs , or MEFs containing MFN null mutations transfected with empty vector control ( Ctrl ) or a GASZ expressing plasmid . Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion .

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E. PEG fusion assays of Su9-GFP and COX4-DsRed labeled wild-type MEFs, or MEFs containing MFN null mutations transfected with empty vector control (Ctrl) or a GASZ expressing plasmid. Bar graph represents averaged percentage of areas with different levels of mitochondrial fusion.


A . Gross morphology of testes from MFN1f / f - Cre compared to their wild - type littermates .

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A. Gross morphology of testes from MFN1f/f-Cre compared to their wild-type littermates.


B . Histological studies of testis sections from wild - type mice or MFN1f / f - Cre littermates at different time points postnatal . Red stars : tubules with spermatocytes .

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B. Histological studies of testis sections from wild-type mice or MFN1f/f-Cre littermates at different time points postnatal. Red stars: tubules with spermatocytes.


C . IHF of PLZF ( day 14 postnatal ) , MILI , MVH , and GASZ / CytC ( day 7 ) of testes sections . Scale bars for PLZF are 50 m and 10 m for others .

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C. IHF of PLZF (day 14 postnatal), MILI, MVH, and GASZ/CytC (day 7) of testes sections. Scale bars for PLZF are 50 m and 10 m for others.


D . EM of testical sections from wild - type neonatal mice or MFN1f / f - Cre littermates . N : nucleus .

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D. EM of testical sections from wild-type neonatal mice or MFN1f/f-Cre littermates. N: nucleus.


E . Histochemistry of respiratory enzymes , COX , NADH , and SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

E. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.


A . Tryptophan - fluorescence time course associated to Na + ( blue ) and K + ( black ) binding , respectively , to detergent - purified apo EAAT1CRYST at pH 7 . 4 . Horizontal and vertical scale bars represent time and ∆ F / F0 , respectively . B . Tryptophan - fluorescence time course associated to L - asp binding in presence of 1 mM ( blue ) and absence ( black ) of Na + , respectively , at pH 9 .

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A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively. B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9. C


C . Na + binding isotherms of EAAT1CRYST at pH 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Na + binding ( green ) . Solid lines represent fits of Hill equation .

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C. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.


A . Na + - induced L - glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K + . Choline ( Ch + ) condition was subtracted to the Na + condition .

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A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.


B . Log - log plots of Na + ( red , n = 19 ) and L - asp ( blue , n = 27 ) KD , as a function of pH at [ Na + ] = 0 . 5 mM . Na + ( n = 10 ) and L - asp ( n = 7 ) titration data at pH 7 . 4 and 9 . 0 , respectively , are also reported in Fig . 1 . Solid lines are fits of straight lines .

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B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.


C . Log - log plots of L - asp KD for EAAT1CRYST - E406386Q ( light blue , n = 24 ) and EAAT1CRYST - Y405385F ( dark blue , n = 18 ) , as a function of pH at [ Na + ] = 0 . 5 mM . .

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C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..


A . Purified EAAT1CRYST takes up L - glutamate in liposomes loaded with K + or Rb + , but not with choline ( Ch + ) , or with Ch + and Ca2 + . Plots depict data as in Fig . 3A .

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A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.


B . Ca2 + binding isotherms of EAAT1CRYST at pH = 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Ca2 + binding ( green ) . Symbols represent average and s . e . m . values of at least three independent experiments . Solid lines represent fits of Hill equation and dashed line is the fit in Fig . 1C ( blue line ) corresponding to Na + binding .

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B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.


C . Log - log plot of L - asp KD as a function of Ca2 + ( n = 13 ) .

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C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).


. C - J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice . Data are expressed as CSS - normalized bacterial

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. C-J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice. Data are expressed as CSS-normalized bacterial counts


A - C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle , measured by NMR or GC - MS . Data are expressed as μg / ml or bin intensity ( arbitrary unit - a . u . ) D - F Bar chart with individual points showing the levels of pyruvate , succinate and lactate in wt and mdx mice receiving vehicle or DFZ , measured by NMR .

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A-C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle, measured by NMR or GC-MS. Data are expressed as μg/ml or bin intensity (arbitrary unit - a.u.) D-F Bar chart with individual points showing the levels of pyruvate, succinate and lactate in wt and mdx mice receiving vehicle or DFZ, measured by NMR. G


A - B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle ( DMSO ) , NaB ( 100 mg kg − 1 / daily ) or DFZ ( 1 . 2 mg kg − 1 / daily ) for 3 weeks using the rotarod and weight test . Bar charts show the latency to fall or drop the weight of wt and dystrophic mice .

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A-B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle (DMSO), NaB (100 mg kg−1/daily) or DFZ (1.2 mg kg−1/daily) for 3 weeks using the rotarod and weight test. Bar charts show the latency to fall or drop the weight of wt and dystrophic mice.


A - I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ .

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A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.


J - K Representative blotting and bar chart with individual points showing the expression and / or phosphorylation of pAKT / AKT and COX2 in the gastrocnemius of the indicated six groups of mice .

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J-K Representative blotting and bar chart with individual points showing the expression and/or phosphorylation of pAKT/AKT and COX2 in the gastrocnemius of the indicated six groups of mice.


A - B Levels of AEA and 2 - AG in plasma samples of wt and mdx mice expressed as pmol mg − 1 of wet tissue weight .

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A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.


C - D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice .

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C-D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice.


E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice . F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH .

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E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice. F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH. D


A - C Bar charts with individual points showing the mRNA expression levels of Ulk , Pink and Becn1 measured in control and mdx mice treated with rimonabant ( 0 . 5 mg kg - 1 ) .

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A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).


D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice . E , F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH .

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D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice. E, F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH. D


A - B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).