After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs , cellular fluorescence was measured by flow cytometry . Unlike control GCN4 sensors that homodimerized independent of Fe - S levels and induced consistent fluorescence , fluorescence derived from the GRX2 Fe - S - dependent sensors decreased in hypoxia , as displayed in representative flow cytometric plots ( left ) and by quantification of percentage of positive cells [ ( cell number in M2 gate ) / ( total cell number ) × 100 ] . Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors ( FLAG - tagged ) in hypoxia ( Hyp ) compared with normoxia ( Norm ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1848 for GCN4 ) . In contrast to consistent fluorescence from control GCN4 sensors , Fe - S - dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1 / 2 ( siISCU ) as compared with control ( siCont ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1790 for GCN4 ) .

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After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs, cellular fluorescence was measured by flow cytometry. Unlike control GCN4 sensors that homodimerized independent of Fe-S levels and induced consistent fluorescence, fluorescence derived from the GRX2 Fe-S-dependent sensors decreased in hypoxia, as displayed in representative flow cytometric plots (left) and by quantification of percentage of positive cells [(cell number in M2 gate)/(total cell number) × 100]. Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors (FLAG-tagged) in hypoxia (Hyp) compared with normoxia (Norm) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1848 for GCN4).In contrast to consistent fluorescence from control GCN4 sensors, Fe-S-dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1/2 (siISCU) as compared with control (siCont) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1790 for GCN4).


GRX2 , but not GCN4 , sensor fluorescence was decreased after transfection of miR - 210 oligonucleotide mimic ( miR - 210 ) as compared with control ( miRC ) ( N = 3 , * * * P = 0 . 0002 for GRX2 ; N = 3 , NS P = 0 . 0913 for GCN4 ) . During hypoxic exposure , GRX2 , but not GCN4 , sensor fluorescence was increased after transfection of an antisense miR - 210 inhibitor ( AS210 ) as compared with control ( ASC ) ( N = 3 , * * P = 0 . 003 for GRX2 ; N = 3 , NS P = 0 . 1194 for GCN4 ) .

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GRX2, but not GCN4, sensor fluorescence was decreased after transfection of miR-210 oligonucleotide mimic (miR-210) as compared with control (miRC) (N = 3, ***P = 0.0002 for GRX2; N = 3, NS P = 0.0913 for GCN4). During hypoxic exposure, GRX2, but not GCN4, sensor fluorescence was increased after transfection of an antisense miR-210 inhibitor (AS210) as compared with control (ASC) (N = 3, **P = 0.003 for GRX2; N = 3, NS P = 0.1194 for GCN4).


After lentiviral delivery of sensor genes , Fe - S - dependent GRX2 sensor fluorescence was decreased in PECAM - positive cells from PH mouse lung ( Hyp + SU5416 ) as compared with control non - PH mouse lung ( Norm + SU5416 , N = 3 , * * * P < 0 . 001 ) .

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After lentiviral delivery of sensor genes, Fe-S-dependent GRX2 sensor fluorescence was decreased in PECAM-positive cells from PH mouse lung (Hyp + SU5416) as compared with control non-PH mouse lung (Norm + SU5416, N = 3, ***P < 0.001).


By electron paramagnetic resonance ( EPR ) spectroscopy ( representative VHL − / − versus control lung ) , Fe - S cluster signal was decreased ( left graph ) in VHL − / − mice lung tissue ( VHL − / − , N = 3 ) as compared with WT control lung tissues ( WT , N = 5 ) , * * * P = 0 . 0003 .

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 O O O O O O O O

By electron paramagnetic resonance (EPR) spectroscopy (representative VHL−/− versus control lung), Fe-S cluster signal was decreased (left graph) in VHL−/− mice lung tissue (VHL−/−, N = 3) as compared with WT control lung tissues (WT, N = 5), ***P = 0.0003.


By EPR , Fe - S - specific signal was decreased ( right graph ) in human PH - diseased lung tissue harvested at lung transplantation ( PH , N = 3 , Supplementary Table S3 ) compared with non - PH control donor lung tissues ( control , N = 3 ) , * P = 0 . 048 .

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

By EPR, Fe-S-specific signal was decreased (right graph) in human PH-diseased lung tissue harvested at lung transplantation (PH, N = 3, Supplementary Table S3) compared with non-PH control donor lung tissues (control, N = 3), *P = 0.048.


B By immunohistochemistry ( IHC ) , ISCU1 / 2 was unchanged in < 100 μm pulmonary vessels of miR - 210 − / − mice exposed to Hyp + SU5416 ( N = 6 ) versus Norm + SU5416 ( N = 7 ) , NS P = 0 . 2833 .

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B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/−mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833.


C Fe - S - dependent Complex I - specific activity was decreased in WT PH mice as compared with control ( N = 7 / group , left bars , * * * P < 0 . 0001 ) , but activity was preserved in the lungs of miR - 210 − / − mice in either condition ( N = 8 / group , right bars ) , NS P = 0 . 3693 .

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C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/−mice in either condition (N = 8/group, right bars), NS P = 0.3693.


D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice ( left bars , * * * P = 0 . 0006 ) , but GLUT1 was unchanged in miR - 210 − / − mice in either condition ( right bars , NS P = 0 . 9967 ) .

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D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice (left bars, ***P = 0.0006), but GLUT1 was unchanged in miR-210−/− mice in either condition (right bars, NS P = 0.9967).


E 3 - nitrotyrosine ( 3 - NT ) was increased in pulmonary vessels of WT PH mice but was reduced in miR - 210 − / − mice in either condition ( N = 6 / group ) ( * * * P < 0 . 0001 , NS P = 0 . 4087 ) .

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E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087).


F In PH conditions , endothelin - 1 was decreased in miR - 210 − / − lung tissue compared with WT tissue ( N = 5 / group ) , * * P = 0 . 007 .

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F In PH conditions, endothelin-1 was decreased in miR-210−/− lung tissue compared with WT tissue (N = 5/group), **P = 0.007.


G PCNA was increased in WT PH pulmonary vessels but was decreased in miR - 210 − / − tissue exposed to either condition ( N = 5 / group ) ( * * P = 0 . 0096 , * P = 0 . 0124 , * P = 0 . 0263 for miR - 210 − / − ) .

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G PCNA was increased in WT PH pulmonary vessels but was decreased in miR-210−/− tissue exposed to either condition (N = 5/group) (**P = 0.0096, *P = 0.0124, *P = 0.0263 for miR-210−/−).


H PCNA was increased in PECAM + endothelial cells from PH versus non - PH mice ( N = 3 / group , * * P = 0 . 0052 ) .

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H PCNA was increased in PECAM+ endothelial cells from PH versus non-PH mice (N = 3/group, **P = 0.0052).


I Unlike WT mice ( black bars ) demonstrating increased right ventricular systolic pressure ( RVSP ) after Hyp + SU5416 ( N = 10 ) versus Norm + SU5416 ( N = 11 ) , hemodynamic dysregulation was significantly alleviated in miR - 210 − / − mice ( white bars , N = 11 ) ( * * * P < 0 . 0001 , * P = 0 . 0258 , * * P = 0 . 0059 ) .

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I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059).


J Compared with WT controls ( N = 9 ) , miR - 210 − / − mice ( N = 8 ) displayed a blunted increase of the Fulton index ( RV / LV + S ) under PH versus baseline conditions ( expressed as a ratio of RV / LV + S under Hyp + SU5416 versus Norm + SU5416 , * P = 0 . 031 ) .

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J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031).


K - M Under PH ( black bars , N = 8 / group ) versus baseline conditions ( white bars , N = 6 / group ) , pulmonary vascular remodeling was alleviated in miR - 210 − / − mice , as visualized via histology ( L ) , and confirmed by decreased % arteriolar muscularization ( K , * * P = 0 . 001 , * * P = 0 . 0045 for miR - 210 − / − , * P = 0 . 0158 ) and decreased vessel wall thickness ( M , * * * P < 0 . 0001 , * * P = 0 . 0086 ) .

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K-M Under PH (black bars, N = 8/group) versus baseline conditions (white bars, N = 6/group), pulmonary vascular remodeling was alleviated in miR-210−/− mice, as visualized via histology (L), and confirmed by decreased % arteriolar muscularization (K, **P = 0.001, **P = 0.0045 for miR-210−/−, *P = 0.0158) and decreased vessel wall thickness (M, ***P < 0.0001, **P = 0.0086).


B , C Intrapharyngeal delivery of miR - 210 mimic increased miR - 210 in whole lung ( * * * P = 0 . 0002 ) ( B ) and in < 100 - μm pulmonary vessels ( * * P = 0 . 007 ) ( C ) .

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B, C Intrapharyngeal delivery of miR-210 mimic increased miR-210 in whole lung (***P = 0.0002) (B) and in < 100-μm pulmonary vessels (**P = 0.007) (C).


D miR - 210 mimic also repressed ISCU1 / 2 levels in those same caliber vessels ( N = 6 / group ) , * * P = 0 . 0022 .

D miR-210 mimic also repressed ISCU1/2 levels in those same caliber vessels (N = 6/group), **P = 0.0022.


E Endothelin - 1 was increased in mouse lung tissue after delivery of miR - 210 mimic ( N = 6 / group ) , * P = 0 . 0368 .

E Endothelin-1 was increased in mouse lung tissue after delivery of miR-210 mimic (N = 6/group), *P = 0.0368.


F - H miR - 210 mimic delivery increased RVSP ( N = 8 / group ) ( F ) and vascular remodeling ( α - smooth muscle actin stain , α - SMA ) , as evidenced by increased percent of muscularized ( < 100 μm ) pulmonary vessels ( G ) and increased medial thickening relative to vessel diameter when compared with miR - Control ( H ) ( N = 6 / group ) . * * P = 0 . 0021 for ( F ) , * * P = 0 . 0072 for ( G ) , * P = 0 . 0335 for ( H ) .

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F-H miR-210 mimic delivery increased RVSP (N = 8/group) (F) and vascular remodeling (α-smooth muscle actin stain, α-SMA), as evidenced by increased percent of muscularized (< 100 μm) pulmonary vessels (G) and increased medial thickening relative to vessel diameter when compared with miR-Control (H) (N = 6/group). **P = 0.0021 for (F), **P = 0.0072 for (G), *P = 0.0335 for (H).


B - D Intravenous delivery of anti - miR - 210 down - regulated miR - 210 expression in whole lung tissue ( N = 7 / group ) , * * * P < 0 . 0001 ( B ) , the pulmonary vasculature as assessed by in situ staining ( N = 7 / group ) , * * * P = 0 . 0003 ( C ) , and PECAM + vascular endothelial cells derived from whole lung tissue ( N = 4 / group ) , * * P = 0 . 0013 ( D ) .

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B-D Intravenous delivery of anti-miR-210 down-regulated miR-210 expression in whole lung tissue (N = 7/group), ***P < 0.0001 (B), the pulmonary vasculature as assessed by in situ staining (N = 7/group), ***P = 0.0003 (C), and PECAM+vascular endothelial cells derived from whole lung tissue (N = 4/group), **P = 0.0013 (D).


E Correspondingly , anti - miR - 210 delivery resulted in preservation of ISCU1 / 2 expression in < 100 - μm pulmonary vessels as compared to anti - miR - Control ( N = 5 / group ) , * * P = 0 . 0011 .

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E Correspondingly, anti-miR-210 delivery resulted in preservation of ISCU1/2 expression in < 100-μm pulmonary vessels as compared to anti-miR-Control (N = 5/group), **P = 0.0011.


F Similarly , ISCU1 / 2 expression was preserved in PECAM + endothelial cells derived from mice treated with anti - miR - 210 ( N = 4 / group ) , * * P = 0 . 0016 .

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F Similarly, ISCU1/2 expression was preserved in PECAM+ endothelial cells derived from mice treated with anti-miR-210 (N = 4/group), **P = 0.0016.


G Endothelin - 1 was decreased in lung tissue from mice treated with anti - miR - 210 during Hyp + SU5416 exposure ( N = 6 / group ) , * P = 0 . 0181 .

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G Endothelin-1 was decreased in lung tissue from mice treated with anti-miR-210 during Hyp + SU5416 exposure (N = 6/group), *P = 0.0181.


H As assessed by in situ immunofluorescence , PCNA was decreased in pulmonary vessels ( < 100 μm ) after anti - miR - 210 delivery , * * P = 0 . 001 .

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H As assessed by in situ immunofluorescence, PCNA was decreased in pulmonary vessels (< 100 μm) after anti-miR-210 delivery, **P = 0.001.


I - K Anti - miR - 210 delivery ( N = 10 / group ) ameliorated the elevation of RVSP , * * P = 0 . 0031 ( i ) and vascular remodeling , as reflected by increased percent of muscularized arterioles ( N = 8 / group , * * P = 0 . 0091 ) ( J ) and increased medial thickness relative to vessel diameter in < 100 - μm pulmonary vessels ( N = 6 / group , * * P = 0 . 0021 ) ( K ) .

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I-K Anti-miR-210 delivery (N = 10/group) ameliorated the elevation of RVSP, **P = 0.0031 (i) and vascular remodeling, as reflected by increased percent of muscularized arterioles (N = 8/group, **P = 0.0091) (J) and increased medial thickness relative to vessel diameter in < 100-μm pulmonary vessels (N = 6/group, **P = 0.0021) (K).


M Pharmacologic inhibition of miR - 210 ( N = 7 / group ) down - regulated pulmonary expression of miR - 210 , * * * P < 0 . 0001 ( 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 O O O O O O

M Pharmacologic inhibition of miR-210 (N = 7/group) down-regulated pulmonary expression of miR-210, ***P < 0.0001 (M).


N Pharmacologic inhibition of miR - 210 ( N = 7 / group ) preserved ISCU1 / 2 expression in < 100 - μm pulmonary vessels , * * P = 0 . 0015 ( N ) .

N Pharmacologic inhibition of miR-210 (N = 7/group) preserved ISCU1/2 expression in < 100-μm pulmonary vessels, **P = 0.0015 (N).


O - Q In contrast to anti - miR - Control ( N = 8 ) , anti - miR - 210 ameliorated the elevation of RVSP ( N = 10 / group , * * * P < 0 . 0001 ) ( O ) and lung remodeling ( N = 7 / group , * * P = 0 . 0022 , * P = 0 . 0167 ) ( P - Q ) .

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O-Q In contrast to anti-miR-Control (N = 8), anti-miR-210 ameliorated the elevation of RVSP (N = 10/group, ***P < 0.0001) (O) and lung remodeling (N = 7/group, **P = 0.0022, *P = 0.0167) (P-Q).


A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416 , intravenous delivery of anti - miR - 210 ameliorated the elevation of RVSP compared with anti - miR - Control ( N = 6 / group , * * * P = 0 . 001 ) .

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A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001).


C , D Consistent with specific delivery to the vascular endothelium after intravenous administration ( Dahlman et al , 2014 ) , 7C1 - mediated delivery of anti - miR - 210 ( N = 6 / group ) decreased miR - 210 expression in PECAM + pulmonary vascular endothelial cells , * * P = 0 . 0013 , ( D ) but not PECAM - negative pulmonary cells , NS P = 0 . 3775 ( C ) .

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C, D Consistent with specific delivery to the vascular endothelium after intravenous administration (Dahlman et al, 2014), 7C1-mediated delivery of anti-miR-210 (N = 6/group) decreased miR-210 expression in PECAM+ pulmonary vascular endothelial cells, **P = 0.0013, (D) but not PECAM-negative pulmonary cells, NS P = 0.3775 (C).


E Correspondingly , endothelial - specific anti - miR - 210 ( N = 6 / group ) increased ISCU1 / 2 expression in PECAM + cells even in the presence of chronic hypoxia , * * * P = 0 . 0003 .

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E Correspondingly, endothelial-specific anti-miR-210 (N = 6/group) increased ISCU1/2 expression in PECAM+ cells even in the presence of chronic hypoxia, ***P = 0.0003.


F - I Delivery of anti - miR - 210 ( N = 5 / group ) decreased endothelin - 1 in hypoxic mouse lung tissue , * P = 0 . 0198 ( F ) . As a result , in contrast to anti - miR - Control ( N = 8 ) , endothelial - specific anti - miR - 210 ameliorated the elevation of RVSP ( N = 6 ) , * * P = 0 . 0024 ( G ) , and pulmonary vascular remodeling ( N = 6 / group ) , * * P = 0 . 0021 , * P = 0 . 0117 ( H - I ) .

F-I Delivery of anti-miR-210 (N = 5/group) decreased endothelin-1 in hypoxic mouse lung tissue, *P = 0.0198 (F). As a result, in contrast to anti-miR-Control (N = 8), endothelial-specific anti-miR-210 ameliorated the elevation of RVSP (N = 6), **P = 0.0024 (G), and pulmonary vascular remodeling (N = 6/group), **P = 0.0021, *P = 0.0117 (H-I).


B , C Pharmacologic inhibition of ISCU ( N = 8 / group ) did not influence miR - 210 expression in the lungs either after Norm + SU5416 , NS P = 0 . 8634 ( B ) or Hyp + SU5416 , NS P = 0 . 9786 ( C ) .

B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5416, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C).


D As compared with control siRNA ( siCont ) where ISCU1 / 2 expression was decreased after exposure to Hyp + SU5416 ( white bars ) , lung delivery of siRNA specific for ISCU1 / 2 ( siISCU ) via Staramine - mPEG nanocomplexes ( Polach et al , 2012 ) down - regulated ISCU1 / 2 in < 100 - μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 ( N = 5 / group , * * * P = 0 . 0006 , * * P = 0 . 0019 for Hyp + SU5416 , * * P = 0 . 0029 for siCont ) .

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D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), lung delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont).


E By electron paramagnetic resonance ( EPR ) spectroscopy ( representative siCont versus siISCU lung ) , Fe - S cluster signal was decreased ( quantitative graph , right ) in siISCU mouselung tissue ( N = 4 mice ) compared with siCont ( N = 5 mice ) , * P = 0 . 0143 .

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E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouselung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143.


F Endothelin - 1 was increased in lung tissue after siISCU delivery under normoxic conditions ( N = 5 / group ) , * * P = 0 . 0027 .

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F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.


G - I In the absence ( left bars ) and presence ( right bars ) of Hyp + SU5416 for 2 weeks , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) and increased pulmonary vascular remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

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G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


G - I In the absence ( left bars ) and presence ( right bars ) of Hyp + SU5416 for 2 weeks , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) and increased lung remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased lung remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


C , Immunoblot analysis of protein lysates from MEF cells treated with EBSS , 250 nM Torin - 1 or 100 μM NaAsO2 for 1h and run under non - reducing conditions . For refeed condition , starved cells were incubated in complete medium for the indicated times .

O O O 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 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

C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1h and run under non-reducing conditions. For refeed condition, starved cells were incubated in complete medium for the indicated times.


C , Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - FLAG - WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non - reducing condition .

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

C, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-FLAG-WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing condition.


D , Immunoblot analysis of protein lysates from ARPE - 19 cells overexpressing TFEB - FLAG or TFE3 - MYC , treated with 250 nM Torin - 1 for 1 h and run under non - reducing condition . E , Quantification of immunoblot data shown in ( D ) . Data are presented as mean ± SD using two - tailed t - test ( * ) p < 0 . 05 from three independent experiments .

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

D, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG or TFE3-MYC, treated with 250 nM Torin-1 for 1 h and run under non-reducing condition. E, Quantification of immunoblot data shown in (D). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 from three independent experiments.


D , Relative quantitative RT - PCR analysis of the mRNA expression of autophagy - ( UVRAG ) , mitochondria - ( PGC - 1α ) and lysosome ( MCOLN1 , ATPV1C1 and HEXA ) - related genes in ARPE - 19 cells overexpressing TFEB - FLAG - WT or TFEB - FLAG - C212A . Data are presented as mean ± SD using two - way ANOVA , ( ns ) not significant as compared to the same gene in TFEB - WT overexpressing cells , from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), mitochondria- (PGC-1α) and lysosome (MCOLN1, ATPV1C1 and HEXA)-related genes in ARPE-19 cells overexpressing TFEB-FLAG-WT or TFEB-FLAG-C212A. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same gene in TFEB-WT overexpressing cells, from three independent experiments.


G , Relative quantitative RT - PCR analysis of the mRNA expression of lysosome - related genes ( MCOLN1 and ATPV61C1 ) in ARPE - 19 and HeLa cells treated with 100 μM and 50 μM NaAsO2 respectively for the indicated times . Data are presented as mean ± SD using one - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 from two independent experiments .

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

G, Relative quantitative RT-PCR analysis of the mRNA expression of lysosome-related genes (MCOLN1 and ATPV61C1) in ARPE-19 and HeLa cells treated with 100 μM and 50 μM NaAsO2 respectively for the indicated times. Data are presented as mean ± SD using one-way ANOVA (*)p<0.05, (**)p< 0.01, (***)p<0.001 from two independent experiments.


D , Relative quantitative RT - PCR analysis of the mRNA expression of autophagy - ( UVRAG ) , lysosome - ( ATP6V1C1 ) , oxidative stress response - ( TXNRD1 and HMOX1 ) , cell cycle control - ( CDKN1a ) and cytokine production ( IL6 ) - related genes in TFEB / TFE3 - double knockout MEFs cells stably expressing TFEB - FLAG - WT or TFEB - FLAG - C212A and treated with 5 μM arsenic trioxide ( ATO ) or 12 . 5 μM NaAsO2 for 12 h . Data are presented as mean ± SD using two - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 , and ( * * * * ) p < 0 . 0001 from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), lysosome- (ATP6V1C1), oxidative stress response- (TXNRD1 and HMOX1), cell cycle control- (CDKN1a) and cytokine production (IL6) -related genes in TFEB/TFE3-double knockout MEFs cells stably expressing TFEB-FLAG-WT or TFEB-FLAG-C212A and treated with 5 μM arsenic trioxide (ATO) or 12.5 μM NaAsO2 for 12 h. Data are presented as mean ± SD using two-way ANOVA (*)p<0.05, (**)p<0.01, (***)p<0.001, and (****)p<0.0001 from three independent experiments.


C , Representative examples of HLH - 30 : : 3xFLAG : : eGFP subcellular localization : Cytoplasmic , diffuse GFP distribution along the whole body of the worm ; Weak nuclear , some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells ; Strong nuclear , distinct nuclear GFP labeling in cells along the worm body , including neurons in the head area . Arrowheads denote nuclear localization of HLH - 30 : : 3xFLAG : : eGFP . Scale bar 200 µm and 50 µm . D , Quantification of the subcellular distribution of HLH - 30 : : 3xFLAG : : eGFP and HLH - 30 ( C284A ) : : 3xFLAG : : eGFP upon treatment with different stimuli . Data are from three independent experiments with at least 50 animals per experiment .

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

C, Representative examples of HLH-30::3xFLAG::eGFP subcellular localization: Cytoplasmic, diffuse GFP distribution along the whole body of the worm; Weak nuclear, some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells; Strong nuclear, distinct nuclear GFP labeling in cells along the worm body, including neurons in the head area. Arrowheads denote nuclear localization of HLH-30::3xFLAG::eGFP. Scale bar 200 µm and 50 µm. D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP upon treatment with different stimuli. Data are from three independent experiments with at least 50 animals per experiment.


A , Lifespan assay of hlh - 30 mutants at 25 ° C . Graph represents Kaplan - Meier survival plots of two independent experiments initiated with 100 animals per strain . ( * * ) p < 0 . 01 and ( * * * * ) p < 0 . 0001 by Log - rank ( Mantel - Cox ) test compared to wild type control . B , Survival of hlh - 30 mutants on 7 . 5 mM NaAsO2 . Data are the mean ± SD of three independent experiments initiated with 120 animals per strain . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control . C , Survival of hlh - 30 mutants to infection by Staphylococcus aureus . Data are the mean ± SD of three independent experiments initiated with 100 animals per strain . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control .

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

A, Lifespan assay of hlh-30 mutants at 25°C. Graph represents Kaplan-Meier survival plots of two independent experiments initiated with 100 animals per strain. (**)p<0.01 and (****)p<0.0001 by Log-rank (Mantel-Cox) test compared to wild type control. B, Survival of hlh-30 mutants on 7.5 mM NaAsO2. Data are the mean ± SD of three independent experiments initiated with 120 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control. C, Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± SD of three independent experiments initiated with 100 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control.


E , Synthetic developmental delay phenotype of eat - 2 ( ad1116 ) worms carrying mutations in the hlh - 30 gene . Data are the mean ± SD of three independent experiments with at least 20 animals per assay . ( * * * * ) p < 0 . 0001 by One - way ANOVA with multiple comparison test . F , Representative images of worms quantified in ( E ) . 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 O O O O O O O O O O O O O O O O O O O O O O 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, Synthetic developmental delay phenotype of eat-2(ad1116) worms carrying mutations in the hlh-30 gene. Data are the mean ± SD of three independent experiments with at least 20 animals per assay. (****)p<0.0001 by One-way ANOVA with multiple comparison test. F, Representative images of worms quantified in (E). Scale bars 200 µm.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO in saline ( PMO - S ) , PMO - G ( IV ) or PMO - G ( D ) ( scale bar = 100μm ) . PMO - G ( IV ) refers to intravenous injection of PMO in combination with intravenous injection of 5 % glycine every other day for 3 weeks . PMO - G ( D ) represents intravenous injection of PMO supplemented with 1 % glycine orally .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( C ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( IV ) , PMO - G ( D ) or PMO - S . 0 . 5µg , 2 . 5 µg , 5µg , 10µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , D - diaphragm and A - abdominal muscle . ( D ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( i . v . , n = 4 ) , PMO - G ( D25 and D50 , n = 3 ) and PMO - S ( n = 3 ) ( * P < 0 . 05 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle. (D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( A ) Muscle function was assessed to determine the physical improvement with grip strength test ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( B ) Measurement of bodyweight changes of DKO mice treated with PMO - G or PMO - S ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( C ) Measurement of TA muscle mass of DKO mice treated with PMO - G or PMO - S ( n = 3 , * P < 0 . 05 ; one - way ANOVA post - hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( D ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) . TA - tibialis

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). TA-tibialis anterior


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( E , F ) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle and D - diaphragm . 2 . 5 µg , 5µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( B ) Measurement of Ejection Fraction ( EF ) and Fractional Shortening ( FS ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 mice ( n = 6 ) with echocardiography ( * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (B) Measurement of Ejection Fraction (EF) and Fractional Shortening (FS) in treated DKO mouse hearts (n=3) and C57BL/6 mice (n=6) with echocardiography (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( C ) Morphological examination of treated DKO mouse hearts with H & E staining ( scale bar = 800μm ) . Boxed areas are magnified . ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (C) Morphological examination of treated DKO mouse hearts with H&E staining (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test) Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Immunohistochemistry for immunoglobin G ( IgG ) in treated DKO mouse hearts ( D , scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Immunohistochemistry for immunoglobin G (IgG) in treated DKO mouse hearts (D, scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Muscle function was evaluated to determine the physical improvement with grip strength ( E ) or running wheel tests ( F ) for treated DKO ( n = 3 ) or C57BL / 6 mice ( n = 4 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Muscle function was evaluated to determine the physical improvement with grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( G ) Immunohistochemistry for dystrophin expression in body - wide muscles from treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (G) Immunohistochemistry for dystrophin expression in body-wide muscles from treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( H ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 20µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (H) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Survival rate of DKO mice treated with PMO - GM ( n = 6 ) . The number of untreated DKO controls is 15 .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Survival rate of DKO mice treated with PMO-GM (n=6).The number of untreated DKO controls is 15.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Measurement of kyphosis index of DKO mice treated with PMO - GM ( scale bar = 5mm ) and wild - type C57BL / 6 mice ( scale bar = 8mm ) ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Measurement of kyphosis index of DKO mice treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of tidal volume in treated DKO mice with a cardio - respiratory gated technique ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of tidal volume in treated DKO mice with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( E - F ) Examination of EF and FS ( E ) and stroke volume and cardiac output ( F ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 ( n = 5 ) with echocardiography ( * P < 0 . 05 , * * P < 0 . 001 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (E-F) Examination of EF and FS (E) and stroke volume and cardiac output (F) in treated DKO mouse hearts (n=3) and C57BL/6(n=5) with echocardiography (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Masson trichrome ' s staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts ( scale bar = 800 μm ) . Boxed areas are magnified ( n = 3 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Masson trichrome's staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( H ) Measurement of serum creatine kinase - MB ( CK - MB ) levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (H) Measurement of serum creatine kinase-MB (CK-MB) levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( I - J ) Muscle function was assessed to determine the physical improvement with grip strength ( I ) or running wheel tests ( J ) in treated DKO ( n = 4 ) and C57BL / 6 mice ( n = 6 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (I-J) Muscle function was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( A ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (A) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 25µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Re - localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture ( scale bar = 50μm ) . The arrowheads point to identical myofibers .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Re-localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of serum CK levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Measurement of serum indices from treated DKO mice to reflect liver ( E ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of serum CK levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Measurement of serum indices from treated DKO mice to reflect liver (E) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . Measurement of serum indices from treated DKO mice to reflect kidney ( F ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. Measurement of serum indices from treated DKO mice to reflect kidney (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Morphological examination of liver and kidney from treated DKO mice ( scale bar = 50 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Morphological examination of liver and kidney from treated DKO mice (scale bar=50 μm).


( I ) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via

(I) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via immunoblot


( M ) Death rates in TNF - treated cell lines including clonal RelA - knockout population ( mean of three independent experiments ± standard deviation ) .

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

(M) Death rates in TNF-treated cell lines including clonal RelA-knockout population (mean of three independent experiments ± standard deviation).


( A ) TNF - induced mRNA expression in indicated L929 cell lines measured via qRT - PCR ( mean of three independent experiments ± standard deviation , two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 ) .

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

(A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.05, **P<0.01).


( B ) Immunoblot after co - immunoprecipitation ( IP ) of RIPK3 . FT , flow through . ( C ) Relative quantification of A20 in RIPK3 - IP fraction ( means and statistical significance established for 0 , 2 , 4 . 5 and 8 hour time points from three independent experiments ± standard deviation ; two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 , n . s . , P > 0 . 05 ) .

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(B) Immunoblot after co-immunoprecipitation (IP) of RIPK3. FT, flow through. (C) Relative quantification of A20 in RIPK3-IP fraction (means and statistical significance established for 0, 2, 4.5 and 8 hour time points from three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01, n.s., P>0.05).


( I , J ) ( I ) TNF - induced distributions of death times ( representative data of three independent experiments ) , and ( J ) death rates obtained by live - cell microscopy ( mean of three independent experiments ± standard deviation ) .

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(I,J) (I) TNF-induced distributions of death times (representative data of three independent experiments), and (J) death rates obtained by live-cell microscopy (mean of three independent experiments ± standard deviation).


( G , H ) ( G ) Distribution of death times ( representative data of three independent experiments ) , or ( H ) death rates in TNF - treated wt , parental A20 KO cells , or A20 KO cells reconstituted with an NFκB - inducible transgene ( fIL8 - A20 ; mean of three independent experiments ± standard deviation ) .

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(G,H) (G) Distribution of death times (representative data of three independent experiments), or (H) death rates in TNF-treated wt, parental A20 KO cells, or A20 KO cells reconstituted with an NFκB-inducible transgene (fIL8-A20; mean of three independent experiments ± standard deviation).


( J ) experimental measurements of 24 - hour fractional survival after varying durations of transient or sustained ( 24h ) TNF stimulation ( mean of three independent experiments ± standard deviation ; two - tailed Student ' s t - test * * * P < 0 . 001 , or no statistically significant difference , n . s . , P > 0 . 05 ) .

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(J) experimental measurements of 24-hour fractional survival after varying durations of transient or sustained (24h) TNF stimulation (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001, or no statistically significant difference, n.s., P>0.05).


( H ) Death rates in TNF - treated indicated cell lines including isogenic IκBα / IκBε - knockout population ( mean of three independent experiments ± standard deviation ) .

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(H) Death rates in TNF-treated indicated cell lines including isogenic IκBα/IκBε-knockout population (mean of three independent experiments ± standard deviation).


( I ) Death rates in TNF - treated cell lines treated with targeting ( siA20 ) or non - targeting ( siCon ) siRNA ( mean of three independent experiments ± standard deviation ) .

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(I) Death rates in TNF-treated cell lines treated with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation).


( a ) Immunoblot for the indicated Cx of homogenates ( Hom ) and autophagic vacuoles ( AV ) isolated from fed or 6 h starved ( Stv ) mice . Quantification of enrichment is shown in Supplementary Fig . 1a .

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(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a.


( b ) Immunofluorescence for LC3 and Atg16 in normal rat kidney ( NRK ) cells expressing GFP - Cx43 maintained in the presence or absence of serum for 4 h . Single channels are shown in reverse black and white , and magnification of single and merged images are shown in colour . Arrows : co - localization of Atg16 / Cx43 ( yellow ) or Atg16 / Cx43 / LC3 ( white ) . Quantification is shown in Supplementary Fig . 1c .

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(b) Immunofluorescence for LC3 and Atg16 in normal rat kidney (NRK) cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4 h. Single channels are shown in reverse black and white, and magnification of single and merged images are shown in colour. Arrows: co-localization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c.


( c ) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum - supplemented media without additions ( None ) or treated with chloroquine ( CQ ) for 4 h . Full fields are shown in Supplementary Fig . 1d .

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(c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d.


( d ) Maximum projection confocal microscopy 3D reconstruction ( left ) followed by a single - plane time - lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and sfGFP - ATG16 .

(d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single-plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and sfGFP-ATG16.


( e ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 but knocked down for Eps15 . Single channels are shown in reverse black and white , and merged images in colour . Inset shows higher magnification . Bottom : Quantification of co - localization between Atg16 and Cx43 ( n = 3 wells , 4 independent experiments , > 20 cells per experiment ) . Values are mean + s . e . m . * P 0 . 05 .

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(e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05.


( f , g ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence ( f , g ) or absence of serum ( 4 h ; g ) . I : input ; FT : flow through .

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(f,g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence (f,g) or absence of serum (4 h; g). I: input; FT: flow through.


( h ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 from untreated WT MEFs , on incubation with 3 - methyladenine ( 3MA ) or in MEFs from Atg5 - null mice ( Atg5 − / − ) .

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(h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−).


( i ) Immunoblot for Flag and Cx43 of immunoprecipitates ( IP ) of endogenous Cx43 in WT MEFs transfected with the indicated Flag - tagged constructs of truncated Atg16 .

(i) Immunoblot for Flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in WT MEFs transfected with the indicated Flag-tagged constructs of truncated Atg16.


( a ) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) grown in serum - supplemented media . Reverse black and white channels ( top ) and colour images ( bottom ) . Right : Average number of LC3 and GABARAP puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(a) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43-null mice (Cx43−/−) grown in serum-supplemented media. Reverse black and white channels (top) and colour images (bottom). Right: Average number of LC3 and GABARAP puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( b ) Electron micrographs of WT and Cx43 − / − MOBs grown in serum - supplemented media . Yellow arrows : autophagic vacuoles . Insets : autophagic vacuoles ( AV ) at higher magnification . Right : Percentage of cytoplasm area occupied by AV ( top ) and number of AV per area ( bottom ; n = 3 mice , > 10 micrographs per mice ) .

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(b) Electron micrographs of WT and Cx43−/− MOBs grown in serum-supplemented media. Yellow arrows: autophagic vacuoles. Insets: autophagic vacuoles (AV) at higher magnification. Right: Percentage of cytoplasm area occupied by AV (top) and number of AV per area (bottom; n = 3 mice, >10 micrographs per mice).


( c ) Immunoblot for LC3 of the same cells grown in serum - supplemented media untreated or treated with lysosomal protease inhibitors ( PI ) for 2 or 4 h . Top : representative immunoblot . GAPDH is shown as loading control . Bottom : LC3 flux ( left ) and increase in LC3 - II during lysosomal inhibition ( right ; n = 3 independent experiments ) .

(c) Immunoblot for LC3 of the same cells grown in serum-supplemented media untreated or treated with lysosomal protease inhibitors (PI) for 2 or 4 h. Top: representative immunoblot. GAPDH is shown as loading control. Bottom: LC3 flux (left) and increase in LC3-II during lysosomal inhibition (right; n = 3 independent experiments).


( d ) Representative images of the same cells transfected with the mCherry - GFP - LC3 tandem reporter and grown in serum - supplemented media . Single - channel images ( inverted black and white ) and merged images ( colour ) . Bottom : Number of vesicles positive for GFP and mCherry ( autophagosomes ; APG ) and only for mCherry ( autophagolysosomes ; APL ; n = 4 wells , 4 independent experiments , > 30 cells per experiment ) .

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(d) Representative images of the same cells transfected with the mCherry-GFP-LC3 tandem reporter and grown in serum-supplemented media. Single-channel images (inverted black and white) and merged images (colour). Bottom: Number of vesicles positive for GFP and mCherry (autophagosomes; APG) and only for mCherry (autophagolysosomes; APL; n = 4 wells, 4 independent experiments, >30 cells per experiment).


( e ) Electron micrographs of livers from WT and Cx43 + / − mice . Arrows indicate AV . Right : Percentage of cytosolic area occupied by AVs ( n = 3 mice , > 10 micrographs per mice ) .

(e) Electron micrographs of livers from WT and Cx43+/− mice. Arrows indicate AV. Right: Percentage of cytosolic area occupied by AVs (n = 3 mice, >10 micrographs per mice).


( f ) Immunofluorescence for LC3 in Cx43 − / − MOBs expressing GFP - tagged full - length Cx43 . Single channels ( inverted black and white ) and merged channels ( colour ) . Asterisks : transfected ( green ) and untransfected ( red ) cells . ( g ) LC3 - positive puncta per cell in the indicated cells ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . All values are mean + s . e . m . and differences from WT were significant for * P 0 . 01 . Nuclei are highlighted with DAPI . Scale bars : 5 μm in fluorescence images ; 1 μm and 0 . 1 μm in top panels and insets of b and e . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(f) Immunofluorescence for LC3 in Cx43−/− MOBs expressing GFP-tagged full-length Cx43. Single channels (inverted black and white) and merged channels (colour). Asterisks: transfected (green) and untransfected (red) cells. (g) LC3-positive puncta per cell in the indicated cells (n = 3 wells, 3 independent experiments, >50 cells per experiment). All values are mean + s.e.m. and differences from WT were significant for *P 0.01. Nuclei are highlighted with DAPI. Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of b and e. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for LC3 in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) treated with vehicle or with 18α glycyrrhetinic acid ( 18α GA ) . Glyceraldehyde - 3 - dehydrogenae ( GPD ) is shown as the loading control . Levels of LC3 - II ( left ) and LC3 - II flux ( right ; n = 3 independent experiments ) .

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(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments).


( b ) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid . Where indicated , chloroquine ( CQ ) was added to block lysosomal degradation . Bottom : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) .

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(b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).


( c ) Immunofluorescence for LC3 in WT or Cx43 − / − MOBs treated with the indicated kinase inhibitors for 2 h . Insets : Higher magnification images in inverted black and white . ( d ) Endogenous LC3 puncta in the same cells shown in c ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . * , differences from WT ; § , differences from untreated .

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(c) Immunofluorescence for LC3 in WT or Cx43−/− MOBs treated with the indicated kinase inhibitors for 2 h. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n = 3 wells, 3 independent experiments, >50 cells per experiment). *, differences from WT; §, differences from untreated.


( e ) LC3 puncta in Cx43 − / − MOBs transfected with constructs coding for different forms of Cx43 ( full length , CT , ΔCT258 or ΔCT245 ) tagged as indicated in the scheme . Left : Representative images . Merged fields ( left ) and LC3 channel in reverse black and white ( right ) are shown ( images of GFP - Cx43 are shown in Fig . 2f ) . Right : Quantification of the average number of LC3 puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(e) LC3 puncta in Cx43−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT258 or ΔCT245) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP-Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( f , g ) Immunoblot for the indicated proteins of the immunoprecipitates ( IP ) of Atg16 from MEFs transfected with GFP - tagged full - length ( FL - ) Cx43 or Flag - tagged C terminus ( CT - ) Cx43 ( f ) or ΔCT258 - Cx43 ( g ) I : input ; FT : flow through . GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control . All values are mean + s . e . m . Differences from the control were significant for ( * , § ) P 0 . 01 and ( * * , § § ) P 0 . 001 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(f,g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full-length (FL-) Cx43 or Flag-tagged C terminus (CT-) Cx43 (f) or ΔCT258-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control. All values are mean + s.e.m. Differences from the control were significant for (*, §)P 0.01 and (**, §§)P 0.001. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblots for the indicated proteins in MEFs treated with tamoxifen ( Tx ) or lindane ( Lind ) in the absence or presence of lysosomal protease inhibitors ( PI ) . Right : Changes in LC3 - II levels relative to those detected in untreated cells ( n = 4 independent experiments ) .

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(a) Immunoblots for the indicated proteins in MEFs treated with tamoxifen (Tx) or lindane (Lind) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n = 4 independent experiments).


( b ) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine ( CQ ) for 3 h . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . ( c ) Time course of changes in the average LC3 - positive puncta per cell after addition of lindane to control NRK cells .

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(b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3 h. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of lindane to control NRK cells.


( d ) Fluorescence images of WT or Cx43 − / − MOBs transfected with mCherry - GFP - LC3 and incubated or not with tamoxifen or lindane . Bottom : Quantification of autophagolysosomes ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(d) Fluorescence images of WT or Cx43−/− MOBs transfected with mCherry-GFP-LC3 and incubated or not with tamoxifen or lindane. Bottom: Quantification of autophagolysosomes (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( e ) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds . Representative images as merged channels are shown . Right : quantification of the average of cytosolic Atg16 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) .

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(e) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds. Representative images as merged channels are shown. Right: quantification of the average of cytosolic Atg16-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).


( f ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 treated with lindane and untreated . Single channel images in inverse black and white and merged colour images are shown . Outlined areas are shown at higher magnification . Arrows : double ( yellow ) and triple ( white ) co - localization .

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(f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 treated with lindane and untreated. Single channel images in inverse black and white and merged colour images are shown. Outlined areas are shown at higher magnification. Arrows: double (yellow) and triple (white) co-localization.


( g , h ) Immunostaining for Atg16 in Cx43 knocked down ( KD ) ( g ) or knocked out ( − / − ) ( h ) cells . Right : quantification of the average of cytosolic Atg16 positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(g,h) Immunostaining for Atg16 in Cx43 knocked down (KD) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).