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

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

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 for beta catenin targets Axin2 Sox9 in uninfected infected Lgr4 - / - 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 ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TNF - α signaling via NF - κB .

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

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 GSII in mouse antrum tissue from uninfected infected Lgr4 - / - control mice . Insets show magnified . Scale bars : 50 µm .

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

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 of p65 + as of total DAPI - stained of uninfected infected Lgr4 - / - control mice ( n = 4 biological replicates per group , n = 20 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 ) .

O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mouse antrum tissue of infected Lgr4 - / - in control mice . Insets show magnified . Scale bars : 50µm . Data information : For G represent findings that were reproduced at least twice in laboratory in independent biological replicates .

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

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 of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in uninfected infected Lgr4 - / - mice ( n = 4 - 5 ) 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 ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mouse antrum tissue in infected Lgr4 - / - in control mice . Scale bars : 50 µm . Data information : For I represent findings that were reproduced at least twice in laboratory in independent biological replicates .

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

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

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

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

O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cxcl2 in 2 - week infected Rspo3 - / - mice ( n = 3 ) versus infected control mice ( n = 4 ) . D qPCR expression level of Cxcl1 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 - for C

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

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 with H . pylori . Immunofluorescence labeling for p65 , GSII KI67 in uninfected infected Rspo3 KI control mice . Scale bars : 50 µm . F of p65 + as of total DAPI - stained of uninfected infected Rspo3 KI control mice ( n = 2 - 3 biological replicates per group , n = 20 per mouse ) . G of GSII + of uninfected infected Rspo3 KI control mice ( n = 2 - 3 biological replicates per group , n = 20 per mouse ) . H of Ki67 as of total DAPI - stained of uninfected infected Rspo3 KI control mice ( n = 2 - 3 biological replicates per group , n = 20 per mouse ) . Data information : For , F , G H data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant one - way ANOVA + Tukey ´ s multiple comparison for F , G 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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

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 control mice . Insets show magnified . Scale bars : 50 µm .

O 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

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 2 - month infected Lgr4 - / - control mice ( top panel ) 2 - month infected Lgr5 - / - control mice ( bottom panel ) . Asterisks indicate nonspecific . Scale bars : 50 µm . B of MPO + in uninfected 2 - month infected Lgr4 - / - control mice ( n = 2 biological replicates per group , n = 5 - 10 FOVs per mouse ) ( left panel ) 2 - month infected Lgr5 - / - 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 ) .

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

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 organoid conditions prior to . A Bright - field of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 200 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 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 O O O O O O O O O O O O O O O O O O O O O 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. 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 organoid conditions prior to . B Immunofluorescence labeling for GSII Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 50 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 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 O 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

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 for C 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

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 organoid conditions prior to . E qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within 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 respective medium conditions ( n = 2 - 3 biological replicates per condition ) . Data information : For E F data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ; t - for E 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 O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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 organoid conditions prior to . A Electrophoretic mobility shift ( EMSA ) SDS - PAGE western blot from whole ( WCL ) lysates from gastric organoids untreated or treated with ADP heptose , maintained either in FM in medium without Rspo , without Wnt Rspo , or + BMP2 . Representative from n = 3 biological replicates . B of EMSA 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 I-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

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 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 gel from antibodies against p52 or p50 . IgG lane serves as a negative 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

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

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

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 of Lgr4 , Lgr5 , Gif , Pgc , Muc6 ( B ) 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 - ) .

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

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

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 Western blots of 293T with ectopically expressed GASZ , NRF1 , MVH , or MitoPLD .

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

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 . Mitochondrial localization was monitored by MitoTracker ( Red ) . Scale bars : 15 m .

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

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

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

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 .

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

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


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

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

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 .

O O O O O O 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. 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 .

O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 with ectopically expressed were extracted for soluble bound fraction examined by Western blots .

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 ectopically expressing GASZ , MitoPLD , AIFM1 , or SIRT3 were treated in presence or absence of Triton X - 100 / or Proteinase K , examined by Western blots .

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

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 of GST - tagged GASZ with FLAG - GASZ produced from 293T .

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

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 . Antibodies for Co - IP ( IP ) or Western blots ( WB ) were listed on left .

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

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 , or 293T co - expressed with GASZ fused with N - or C - terminal half of YFP . Ctrl : empty vector control .

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

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

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

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 . Scale bar : 15 m .

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

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

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

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

O O O 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

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

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 of C18 - 4 ( determined from 100 in experiment ) with different types of mitochondria determined by MitoTracker ( Red ) when transfected with Su9 - GFP or GFP - GASZ .

O O O O O O O O O O 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

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 . n = 5 per genotype .

O O O O O O O O O O O O O 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. 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 , SDH on testical sections from mice 10 days postnatal . Scale bar : 100 m .

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

G. 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 MFN - HA in 293T ( A )

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

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


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

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

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 .

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

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

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

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 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 of areas with different of mitochondrial fusion .

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

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 of testes from MFN1f / f - Cre compared to their wild - type .

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

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 at different time points postnatal . Red stars : tubules with spermatocytes .

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. 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 , GASZ / CytC ( day 7 ) of testes sections . Scale bars for PLZF are 50 m 10 m for others .

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

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 . N : nucleus .

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

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


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

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

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 presence ( pink ) absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Na + binding ( green ) . Solid lines represent fits of Hill equation .

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

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 Na + condition .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ( blue , n = 24 ) EAAT1CRYST - Y405385F ( blue , n = 18 ) , as a function of pH at [ Na + ] = 0 . 5 mM . .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 + Ca2 + . Plots depict data as in Fig . 3A .

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

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

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

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 ) .

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

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


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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 points showing mRNA expression of indicated genes measured in gastrocnemius of control mdx mice treated with or without NaB DFZ .

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 bar chart with points showing expression / or phosphorylation of pAKT / AKT COX2 in gastrocnemius of indicated six groups of mice .

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

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

O 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 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 points showing mRNA expression of CB1 CB2 measured in gastrocnemius of indicated groups of mice .

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

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 expression of CB1 CB2 in gastrocnemius of indicated groups of mice . F of CB1 CB2 to housekeeping GAPDH .

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

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 points showing mRNA expression of Ulk , Pink Becn1 measured in control mdx mice treated with rimonabant ( 0 . 5 mg kg - 1 ) .

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

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 expression of LC3I LC3II in gastrocnemius of indicated groups of mice . E , F of LC3I LC3II to housekeeping GAPDH .

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

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 points showing mRNA expression of Il6 Cox2 in control ( vehicle , DMSO ) / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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

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).


A - F Bar chart with points showing mRNA expression of Cb1 , Daglα , Daglβ , Magl , Napepld , Faah in control ( vehicle , DMSO ) / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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

A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah 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).


G - H of AEA 2 - AG were measured in C2C12 exposed to LPS ( 1 μg / ml ) or NaB ( 3 mM ) for 24 h . I Effect of ACEA ( 1 μM ) rimonabant ( 1 μM ) on autophagosome formation measured in C2C12 .

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

G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h. I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells. D


C - J Bar chart with points showing expression of selected miRNAs in control Gpr109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in presence or absence of either NaB ( 3 mM ) , MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) . NaB was also tested in presence or absence of either rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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

C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).


A - E Bar chart showing mRNA expression of IL6 , COX2 , ULK 1 , ATG13 ATG4 mRNA in human myoblasts isolated from one healthy donor ( HD ) five DMD donors ( D1 - D5 ) .

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

A-E Bar chart showing the mRNA expression levels of IL6, COX2, ULK 1, ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor (HD) and five DMD donors (D1- D5).


( A - B ) Hemoglobin ( A ) hematocrit ( B ) of healthy subjects cachectic cancer presenting a loss superior to 10 % of initial ( 19 healthy subjects , 17 cachectic ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(A-B) Hemoglobin (A) and hematocrit (B) levels of healthy subjects and cachectic cancer patients presenting a body weight loss superior to 10% of initial body weight (19 healthy subjects, 17 cachectic patients). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 mRNA in muscle biopsies from cancer - of late stage cachexia ( 19 healthy subjects , 17 cachectic with at least 10 % total loss ) Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C) TFR1 mRNA levels in muscle biopsies from cancer-patients of late stage cachexia (19 healthy subjects, 17 cachectic patients with at least 10% total body weight loss) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) TFR1 mRNA in gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet ( IDD ) combined to a phlebotomy ( PHL ) ( n = 5 - 6 ) Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(F) TFR1 mRNA levels in the gastrocnemius of mice subjected to iron deprivation by feeding with an iron deficient diet (IDD) combined to a phlebotomy (PHL) (n=5-6) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) Cross - sectional of skeletal muscle fiber transfected with shSCR ( scramble ) shTFR1 ( n = 3 - 4 ) representative picture of a shTFR1 transfected fiber . Scale bar = 50µm . ( H ) Cross - sectional of skeletal muscle transfected with TFR - pHuji ( n = 4 ) representative picture of a TFR - pHuji transfected fiber . Scale bar = 50µm . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(G) Cross-sectional area of skeletal muscle fiber transfected with shSCR (scramble) and shTFR1 (n=3-4) and representative picture of a shTFR1 transfected fiber. Scale bar=50µm. (H) Cross-sectional area of skeletal muscle fibers transfected with TFR-pHuji (n=4) and representative picture of a TFR-pHuji transfected fiber. Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( L ) Reprensentative pictures diameter measurements of human myoblast - derived myotubes after 48h treatment with DFO 100µM ( n = 3 ) . Scale bar = 50µm . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(L) Reprensentative pictures and diameter measurements of human myoblast-derived myotubes after 48h treatment with DFO 100µM (n=3). Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C ) TFR1 expression densitometric in mouse gastrocnemius ( n = 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C) TFR1 protein expression and densitometric quantification in mouse gastrocnemius (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( E ) Representative Western blot of IRP2 in mouse gastrocnemius densitometric ( n = 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(E) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Representative Western blot of ferritin Heavy chains in mouse gastrocnemius densitometric ( n = 4 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(H) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I - J ) ICP - MS of total ( I ) - bound ( J ) iron in mouse quadriceps ( n = 4 - 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

(I-J) ICP-MS quantification of total (I) and protein-bound (J) iron in mouse quadriceps (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) ICP - MS of mitochondrial iron in mouse quadriceps ( n = 6 - 8 ) . ( B ) Gastrocnemius heme content quantified by heme ( n = 4 - 6 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(A) ICP-MS quantification of mitochondrial iron in mouse quadriceps (n=6-8). (B) Gastrocnemius heme content quantified by fluorescent heme assay (n=4-6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( C - D ) mRNA of mitochondrial iron importer MFRN2 ( C ) rate limiting enzyme of heme synthesis ALAS2 ( D ) normalized to 18s in mouse gastrocnemius ( n = 4 - 5 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(C-D) mRNA levels of mitochondrial iron importer MFRN2 (C) and the rate limiting enzyme of heme synthesis ALAS2 (D) normalized to 18s in mouse gastrocnemius (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) Succinate Dehydrogenase activity in transversal sections of mouse gastrocnemius corresponding ( n = 3 - 4 ) . Scale bar = 50µm . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

O O O B-MEASURED_VAR I-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

(F) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification (n=3-4). Scale bar= 50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( B ) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM ferric citrate . ( n = 3 )

O O 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) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3)


( G - H ) Representative microscopic pictures diameter of C2C12 myotubes stained for myosin heavy chain ( G ) or human myoblast - derived myotubes ( H ) after 48h treatment with C26 CM ferric citrate ( n = 3 per condition ) . Scale bars = 50 100 µm , respectively . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 CM - treated group .

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

(G-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H)after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.


( B ) Kaplan - Meier depicting survival of C26 - bearing mice after I . V . injection of saline or iron every 5 days post C26 - injection ( 3 - month - old Balb / C , n = 8 - 11 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by chi - square for survival curves Data are mean ± . ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier depicting the survival of C26-tumor bearing mice after I.V. injection of saline or iron every 5 days post C26-injection (3-month-old Balb/C, n=8-11). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by chi-square test for the survival curves Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


Immunofluorescent of myosin heavy chain fast ( green ) slow ( red ) of transversal sections of gastrocnemius ( midbelly ) with corresponding frequency distribution Scale bar = 100µm . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction Data are mean ± . ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

Immunofluorescent staining of myosin heavy chain fast (green) and slow (red) of transversal sections of gastrocnemius (midbelly) with corresponding frequency distribution Scale bar=100µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( I - K ) mRNA of Murf 1 ( I ) , Atrogin 1 ( J ) , REDD1 ( K ) normalized to GAPDH in gastrocnemius ( n = 5 - 14 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by one - way Anova with Bonferroni ' s correction , Data are mean ± . ∗ p < 0 . 05 ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(I-K) mRNA levels of Murf 1 (I), Atrogin 1 (J), and REDD1 (K) normalized to GAPDH in gastrocnemius (n=5-14). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction, Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( B ) Aconitase activity of quadriceps lysates normalized to content ( n = 3 - 7 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(B) Aconitase activity of quadriceps lysates normalized to protein content (n=3-7). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( E ) Representative Western blot densitometric of phospho - AMPK total AMPK ( stripped re - blotted ) in gastrocnemius of C26 - bearing mice after iron carboxymaltose supplementation ( n = 6 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Western blot and densitometric quantification of phospho-AMPK and total AMPK (stripped and re-blotted) in the gastrocnemius of C26-tumor bearing mice after iron carboxymaltose supplementation (n=6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( F - H ) mRNA of ACOT 1 ( F ) , MCD ( G ) , CPT1B ( H ) normalized to GAPDH in gastrocnemius ( n = 5 - 10 ) . Data information : For data , n represents number of biological replicates . Statistical significance was calculated by unpaired , two - Student ' s T - . Data are mean ± . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 compared to control # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 compared to C26 untreated group .

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

(F-H) mRNA levels of ACOT 1 (F), MCD (G), and CPT1B (H) normalized to GAPDH in gastrocnemius (n=5-10). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.


( A - B ) Grip force of dominant or non - dominant arm in iron - deficient cancer , expressed in of baseline force ( A ) absolute values normalized to height ( B ) before / after single dose of iron carboxymaltose ( 15mg / kg , 7 subjects ) . Data information : For data , n represents In graph ( A ) , boxes represent range of values with median value being central band whiskers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Grip force of dominant or non-dominant arm in iron-deficient cancer patients, expressed in percentage of baseline force (A) and absolute values normalized to height (B) before/after single dose of iron carboxymaltose (15mg/kg, 7 subjects). Data information: For all data, n represents In graph (A), the boxes represent the range of values with the median value being the central band and whiskers the SEM.


Overall structure of RBD - ACE2 - B0AT1 . ( A ) Cryo - EM map of RBD - ACE2 - B0AT1 . Left : Overall reconstruction of ternary at 2 . 9 Å . Inset : focused refined map of RBD .

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

Overall structure of the RBD-ACE2-B0AT1 complex.(A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. Left: Overall reconstruction of the ternary complex at 2.9 Å. Inset: focused refined map of RBD.


Overall structure of RBD - ACE2 - B0AT1 . is colored by subunits , with protease domain ( PD ) Collectrin - like domain ( CLD ) colored cyan blue in one of ACE2 protomers , respectively . glycosylation moieties are shown as sticks .

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

Overall structure of the RBD-ACE2-B0AT1 complex. The complex is colored by subunits, with the protease domain (PD) and the Collectrin-like domain (CLD) colored cyan and blue in one of the ACE2 protomers, respectively. The glycosylation moieties are shown as sticks.


Cryo - EM density of interface between RBD ACE2 . density , shown as pink meshes , is contoured at 12 σ .

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

Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.


A , Misreading ( A ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes ( n = 8 misreading , mutant A1555G mitohybrid ribosomes ( n = 8 misreading , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . Mistranslation was determined by assessing misreading at His245 ( CAC codon ) of F - luc gene that was replaced by near - cognate codon CGC or non - cognate codon AGA , both encoding for Arg . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


read - through ( B ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes n = 4 read - through ) , mutant A1555G mitohybrid ribosomes n = 4 read - through ) , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . To determine read - through Asp357 ( GAC codon ) of F - luc gene was replaced by nonsense TGA stop codon . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

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

read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes n=4 read-through), mutant A1555G mitohybrid ribosomes n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


C of 35S - Cysteine 35S - Methionine incorporation by in - vitro translation using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , MT - CO1 mRNA . Following immunoprecipitation , MT - CO1 band was quantified by autoradiography , wild - type mitohybrid ribosomes were set as 1 ( n = 3 ) , error bars indicate ; * * * * p < 0 . 001 ( Student ' s t - ) .

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

C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test).


D of 35S - Cysteine 35S - Methionine incorporation by in - vitro translation in presence of tobramycin using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , MT - CO1 mRNA ; MT - CO1 band was quantified by autoradiography .

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

D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography.


E Misreading of wild - type mitohybrid ribosomes mutant A1555G mitohybrid ribosomes in presence of tobramycin . Misreading was assessed by dual luciferase , results derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity .

O O O O O O O O O O O O O O O O O O O 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 Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.


F In - vitro translation of MT - CO1 - TGA mRNA MT - CO1 - AGA mRNA using mutant A1555G mitohybrid ribosomes in presence of tobramycin ( 0 - 20 µM ) . Autoradiography of immunoprecipitated MT - CO1 ; top : 35S - Met labelling , bottom : 14C - Lys labelling . In - organello translated 35S - Met labelled MT - CO1 was used as a marker . * MT - CO1 , * * MT - CO1 extended by read - through , MT - CO1 AGA - polyA MT - CO1 TGA - polyA constructs used for in - vitro translation are schematized .

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

F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized.


G of 14C - Lys 35S - Met labelled immunoprecipitated MT - CO1 . In - vitro translation using MT - CO1 - TGA mRNA , MT - CO1 - AGA mRNA , mutant A1555G mitohybrid ribosomes in presence of tobramycin ( 0 - 20 µM ) . corresponding MT - CO1 band was quantified by autoradiography .

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

G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.


A Autoradiography of in - organello mitochondrial translation products derived from HEK293 wild - type in presence of tobramycin , incorporation of 35S - Met 35S - Cys in MT - CO1 . Following in - organello translation were immunoprecipitated analyzed by autoradiography . Lane 1 , 3 : controls ; lanes 2 , 4 : in - organello translation in presence of 1000 µM tobramycin . MT - CO1 bands were quantified of 35S - Cys / 35S - Met calculated , 35S - Cys / 35S - Met in absence of tobramycin was set as 1 ( n = 3 ) , error bars indicate .

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

A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.


B Ratios of 35S - Cys / 35S - Met labelled MT - CO1 MT - CO2 synthesized in in - organello translation effect of tobramycin . Following in - organello translation in presence of tobramycin ( 1000 µM , 2000 µM ) were immunoprecipitated analyzed by autoradiography . MT - CO1 MT - CO2 bands were quantified of 35S - Cys / 35S - Met incorporation calculated ( n = 3 ) , error bars indicate ; * p < 0 . 05 , ns , not significant ( Student ' s t - ) .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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 Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).


of 35S - Cys 35S - Met labelled MT - CO1 synthesized in in - organello translation using mitochondria from HEK293 MRPS5 wild - type HEK MRPS5 mutant V336Y . Following in - organello translation , were immunoprecipitated analyzed by autoradiography . MT - CO1 bands quantified ratios calculated . of MRPS5 wild - type was set as 1 ( n = 8 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - ) .

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

Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


D Autoradiography of in - organello mitochondrial translation products derived from HEK293 MRPS5 wild - type in presence of tobramycin ( 0 - 750 µM ) . were 35S - Met labeled loaded either directly on SDS - PAGE ( total , top ) or following poly - lysine immunoprecipitation ( bottom ) . * MT - CO1 * * MT - CO1 with poly - lysine extension .

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

D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.


E of MT - CO1 in HEK293 MRPS5 wild - type HEK293 MRPS5 mutant V336Y . In - organello mitochondrial translation in presence of 35S - Met 750 µM tobramycin . were loaded either directly on SDS - PAGE ( total ) or following poly - lysine immunoprecipitation analyzed by autoradiography . For total samples MT - CO1 band was quantified , for poly - lysine immunoprecipitated samples extended MT - CO1 band was quantified of extended MT - CO1 / total MT - CO1 calculated . of MRPS5 wild - type was set as 1 ( MRPS5 wild - type , n = 5 clones , MRPS5 V336Y , n = 7 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - ) .

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

E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


A Generation time ( n = 8 clones , ± SD ) .

A Generation time (n=8 clones, ± SD).


B Whole in - vivo mitochondrial translation as determined by 35S - Met incorporation ( n = 4 clones , ± ) .

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

B Whole cell in-vivo mitochondrial translation as determined by 35S-Met incorporation (n=4 clones, ± SEM).


C Autoradiography of in - organello translation using 35S - Met labelling .

C Autoradiography of in-organello translation using 35S-Met labelling.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type mutant mouse cortex . A OCR in brain mitochondria from 19 - months mutant mice ( black circle ) compared with OCR from wild - type mice ( circle ) . sequential injection of mitochondrial substrates inhibitors is indicated by dotted lines Values corresponding to different respiratory states 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 O O O O O O O O O O O O O O

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. A OCR in brain mitochondria from 19-months mutant mice (black circle) compared with OCR from wild-type mice (white circle). The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type mutant mouse cortex . Baseline OCR , effect of ( p < 0 . 0001 ) genotype ( p < 0 . 05 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. Baseline OCR, effect of age (p<0.0001) and genotype (p<0.05).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type mutant mouse cortex . C State 3 OCR ( after addition of ADP ) , effect of ( p < 0 . 001 ) genotype ( p < 0 . 0001 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. C State 3 OCR (after addition of ADP), effect of age (p<0.001) and genotype (p<0.0001).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type mutant mouse cortex . D State 3 uncoupled OCR ( state 3u after addition of FCCP ) , effect of genotype ( p < 0 . 0001 ) - genotype interaction ( p < 0 . 0001 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. D State 3 uncoupled OCR (state 3u after addition of FCCP), effect of genotype (p<0.0001) and age-genotype interaction (p<0.0001).


E Ratio of mitochondrial complex IV and complex II activities.


A Average to center as measure of anxiety in open field ( ANOVA : genotype F1 , 24 = 16 . 24 p = 0 . 0005 , F2 , 24 = 5 . 217 p = 0 . 0131 ) . Mutants show increased anxiety at 9 - 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Average distance to center as measure of anxiety in the open field (ANOVA: genotype F1,24=16.24 p=0.0005, age F2,24=5.217 p=0.0131). Mutants show increased anxiety levels at 9- and 14-months.


B Estimated frequency of rearing activity as measure of exploratory drive in open field ( ANOVA : genotype F1 , 24 = 39 . 40 p < 0 . 0001 , x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . Mutants showed little exploratory activity at 9 - 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.


C - transition , % time spent in bright compartment ( ANOVA : genotype F1 , 16 = 7 . 393 p = 0 . 0152 ) . Reduced scores at 9 - months old indicate increased anxiety .

O O O O O O O O O O O O O O O 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 Light-dark transition test, % time spent in the bright compartment (ANOVA: genotype F1,16=7.393 p=0.0152). Reduced scores at 9-months old animals indicate increased anxiety.


D Exploratory corner visits in phase during adaptation to IntelliCage ( ANOVA : phase F1 , 18 = 617 . 2 p < 0 . 0001 ) . Corner visits during phase as a correlate of exploratory activity showed premature reduction in 9 - months old .

O O O O O O O O O O O O O O O O O O O O O O O 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 Exploratory corner visits in dark and light phase during adaptation to the IntelliCage (ANOVA: phase F1,18=617.2 p<0.0001). Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9-months old animals.


E Learning in hippocampus - dependent IntelliCage serial reversal task ( ANOVA : day F2 , 56 = 26 . 71 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

O O O O O O O O O O O O O O O O O O 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 Learning in the hippocampus-dependent IntelliCage serial reversal task (ANOVA: day F2,56=26.71 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


F Learning in hippocampus - dependent IntelliCage chaining task ( ANOVA : day F2 , 54 = 450 . 1 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

F Learning in the hippocampus-dependent IntelliCage chaining task (ANOVA: day F2,54=450.1 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


G Water - maze place navigation , swim path during acquisition reversal ( ANOVA : day F4 , 104 = 21 . 14 p < 0 . 0001 , genotype F1 , 26 = 13 . 11 p = 0 . 0013 , x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . groups learned : controls showed reduced performance with increasing , mutants showed poor performance already at 9 - 14 - months .

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

G Water-maze place navigation, swim path during acquisition and reversal (ANOVA: day F4,104=21.14 p<0.0001, genotype F1,26=13.11 p=0.0013, age x genotype F2,26=8.204 p=0.0017). All groups learned: controls showed reduced performance with increasing age, mutants showed poor performance already at 9- and 14-months.


H Number of wall approaches as indicator of poor coping with non - spatial strategy choice ( ANOVA : genotype F1 , 26 = 18 . 31 p = 0 . 0002 , F2 , 26 = 4 . 486 p = 0 . 0212 , x genotype F2 , 26 = 7 . 068 p = 0 . 0035 ) . Number of wall approaches increased with in controls was prematurely increased in mutants at 9 - 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Number of wall approaches as indicator of poor coping with the test and non-spatial strategy choice (ANOVA: genotype F1,26=18.31 p=0.0002, age F2,26=4.486 p=0.0212, age x genotype F2,26=7.068 p=0.0035). Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9- and 14-months.


Water - maze probe trial , spatial retention score for target zones 1 - 3 comprising 5 , 10 10 % of pool surface versus remaining 75 % of surface ( ANOVA : zone F3 , 78 = 36 . 04 p < 0 . 0001 , genotype x zone F3 , 78 = 7 . 807 p = 0 . 0001 ) . Spatial selectivity declined with in controls was prematurely reduced in mutants at 9 - 14 - months .

Water-maze probe trial, spatial retention score for target zones 1-3 comprising 5, 10 and 10% of the pool surface versus remaining 75% of surface (ANOVA: zone F3,78=36.04 p<0.0001, genotype x zone F3,78=7.807 p=0.0001). Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9- and 14-months.


Noise exposure ABR measurements . Exposure of mice to broadband noise ( 2 - 10 kHz ) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds . Thresholds in Mrps5V338Y / V338Y mutant mice ( black bars ) were significantly higher after exposure than thresholds in ( open bars ) , ( p < 0 . 0001 , two - way ANOVA for combined 108 110 dB data ) . Frequencies of 12 , 24 32 kHz were selected for measurement to cover mid - frequency to high - frequency regions of cochlea . n = 6 for baseline ; n = 6 for baseline mutants ; n = 3 for noise exposures .

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

Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.


B . ITC binding assays of UVR8 HY5 VP - peptides versus COP1 WD40 domain . top panel represents heats detected during each injection . bottom panel represents integrated heats of each injection , fitted to a one - site binding model ( solid ) . following concentrations were typically used ( titrant into ) : UVR8 - COP1 ( 2500 μM in 175 μM ) ; HY5 - COP1 ( 1500 μM in 175 μM ) . insets show dissociation constants ( Kd ) stoichiometries of binding ( N ) ( ± standard deviation ) .

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

B. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation).