human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells ( B ) were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , the cells were imaged immediately ( starved ) , after which either amino acids ( R + L ) or insulin were added . Thirty minutes later the cells were imaged again . Finally , the mTOR inhibitor , Torin1 was added , and after an additional 30 minutes , the cells were imaged for the last time . Histograms represent changes in the fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin and amino acids significantly increased the a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test


C Raptor - depleted , WT cortical neurons were imaged by 2P - FLIM before and 30 minutes after treatment with insulin . Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin . Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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C Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum - starved in Hank ' s balanced salt solution ( HBSS ) for 2 hours . Next , amino acids ( R + L ) , insulin , insulin + R + L , or the oxidative phosphorylation inhibitor , NaN3 , were added , or the HBSS was replaced with complete medium . One hour later cellular reactive oxygen species ( ROS ) , ATP levels and oxygen consumption were measured . The data were collected from 3 independent assays in which each experimental condition contained 6 - 8 replicates . Error bars represent ± s . e . m . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test


B MP - PAM imaging of wild type ( WT ) mouse cerebral cortex through an open - skull window 1 hour after topical application of amino acids ( R + L ) . A decrease in blood oxygenation of the cortical vasculature was observed , indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity . Data were obtained from 3 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) and cerebral metabolic rate of oxygen ( CMRO2 ) . Error bars represent ± s . e .

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B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m


C MP - PAM imaging of WT mouse cerebral cortex through an open - skull window beginning at baseline , which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin . Baseline levels of O2 saturation and blood flow speed were recorded , after which R + L was applied to the open skull window . One hour later , O2 saturation and blood flow speed were recorded again . Data were obtained from 4 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) and cerebral metabolic rate of oxygen ( CMRO2 ) . Note that no significant changes in O2 saturation or OEF were observed after R + L stimulation , in contrast to what was observed in the absence of rapamycin ( Fig 3B ) . Error bars represent ± s . e .

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C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors ( Control ) , or otherwise identical cells expressing Flag - Raptor fused to either the plasma membrane targeting signal of H - Ras or the lysosomal targeting signal of Rheb were serum - starved in Hank ' s balanced salt solution for 2 hours , and then imaged by 2P - FLIM before and 30 minutes after addition of insulin . Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t - test ( ns : not significant ) . Expression of the Flag - Raptor fusion proteins was confirmed by western blotting ( right panel ) . Note that NiMA was supported when mTORC1 was targeted to lysosomes ( Flag - Raptor Rheb15 ) , but not to the plasma membrane ( Flag - Raptor H - Ras25 ) . Western blots are representative of 3 independent

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 hours, and then imaged by 2P-FLIM before and 30 minutes after addition of insulin. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of 3 independent assays


B Mouse cortical neurons starved in Hank ' s balanced salt solution for 2 hours were treated with AβOs , insulin or amino acids ( R + L ) for 30 minutes , and then were fixed and double labeled with anti - mTOR , and either anti - LAMP1 or Mitotracker CMXRos . Error bars represent ± s . e . m . Data are representative of 3 independent assays

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B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays


C Reducing Bcl - xL expression in WT mouse cortical neurons does not affect NiMA . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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C Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3 - hour pulse of the thymidine analog , EdU . Cells were labeled additionally with the mitochondrial marker , Mitotracker CMXRos , and with an antibody to the neuron marker ,

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A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2


B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~ 60 % , an effect that was blocked by AβOs . Error bars represent ± s . e . m . Statistical analyses were performed using Student " s two - tailed unpaired t - test and are representative of 3 independent

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B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent a


C WT mouse cortical neurons expressing either Flag - Raptor H - Ras25 or Flag - Raptor Rheb15 were serum - starved in AβO - containing Hank " s balanced salt solution for 2 hours . Next , the cells were pulse - labeled for 3 hours with EdU . Note that forcing mTORC1 to the plasma membrane ( Flag - Raptor H - Ras25 ) yielded an ~ 80 % increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin + R + L . In contrast , EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes ( Flag - Raptor - Rheb15 ) . Error bars represent ±

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C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±


A Unmodified ReNcell VM human neuronal progenitor cells were co - plated with comparable Ren - GFP or Ren - APPSL cells into 3 - dimensional Matrigel matrices , and differentiated into neurons for 60 days . Cytosolic GFP is produced by both Ren - GFP and Ren - APPSL cells , and the latter also produce pathogenic human APP with the Swedish and London mutations , and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation , 2P - FLIM was used to monitor NiMA in the ReNcell VM neurons , which were discriminated from Ren - GFP and Ren - APPSL neurons because they lack GFP . Note that NiMA was observed in ReNcell VM neurons when they were co - cultured with Ren - GFP neurons , but not when they were co - cultured with Ren - APPSL neurons . Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines of the same color refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures . Subsequent AβO exposure for 16 hours blocked NiMA , but expression of Flag - Raptor - Rheb15 , which forces mTORC1 to lysosomes , prevented NiMA inhibition by AβOs . Each colored line refers to a single field of view containing 5 - 15 cells , and each identically colored pair of solid and dotted lines refers to the same field of view . Flag - Raptor - Rheb15 expression was confirmed by western blot ting . Statistical analyses were performed using Student ' s two - tailed unpaired t - test . Western blots are representative of 3 independent

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B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blotting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays


A Cortical mouse neurons from tau knockout ( KO ) mice were serum - starved in Hank ' s balanced salt solution for 2 hours . The cells were then imaged for NiMA , after which insulin or amino acids were immediately added , and the cells were then imaged again 30 minutes later Data information : Each colored lin refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Cortical mouse neurons from tau knockout (KO) mice were serum-starved in Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin or amino acids were immediately added, and the cells were then imaged again 30 minutes later Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum - starved in AβO - containing Hank ' s balanced salt solution for 2 hours . The cells were then imaged for NiMA , after which insulin was immediately added , and the cells were then imaged again 30 minutes later . Note that NiMA occurred in the absence or presence of AβOs , which inhibited NiMA in wild type human neuron Data information : Each colored lin refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids ( R + L Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein , a downstream target of the mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein , a downstream target of the mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


( D Human fibroblasts deficient in Tsc2 ( TSC2 Δexons1 - 14 ) were imaged before and 30 minutes after insulin addition . Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or amino acids , and were imaged before and 30 minutes after stimulation . Each colored line refers to a single field of view containing 2 - 5 cells , and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student ' s two - tailed unpaired t -

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(D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test


( E ) Re - expression of human Tsc2 rescues mitochondrial responses to insulin . Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or amino acids , and were imaged before and 30 minutes after stimulation . Each colored line refers to a single field of view containing 2 - 5 cells , and each pair of solid and dotted lines of the same color refers to the same field of view . Re - expression of the Flag - WT Tsc2 was corroborated by indirect immun o fluorescence ( IF ) . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immunofluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test


A . Immunofluorescence microscopy of control , parental and patient fibroblasts labeled for LAMP - 1 . Patient fibroblasts ( VPS41S285P / R662 * and VPS41c . 1423 - 2A > G / R662 * ) show more LAMP - 1 puncta , which are distributed throughout the cell . ( A ' ) Quantification shows a significant increase in the number of LAMP - 1 positive compartments for both patients . > 15 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.


D . HeLaVPS41KO cells transfected with VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 constructs labeled for LAMP - 1 and V5 immunofluorescence microscopy . All VPS41 variants co - localize with LAMP - 1 , indicating that VPS41S285P and VPS41R662 * are recruited to late endosomes / lysosomes . Scale bars 10µm ; zoom of squared area , 1µm .

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D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.


H . VPS41WT / WT , VPS41WT / R662 * and VPS41S285P / R662 * primary fibroblasts incubated with Dextran - 568 for 0 . 5 , 2 , 5 , 8 and 24 hours . Colocalization of Dextran - 568 and SiR - Lysosome Cathepsin D ( SiR - Lyso ) reveals a delay in lysosomal delivery in maternal ( VPS41WT / R662 * ) and patient ( VPS41S285P / R662 * ) cells at 2 hours of Dextran uptake . After 5 hours , maternal cells show similar to control colocalization levels . Patient cells show only after 24 hours colocalization levels similar to control , indicating a delay rather than a block in late endosome - lysosome fusion . > 10 Cells per cell line were quantified .

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

H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.


A . Western blot of LC3 expression levels in control and patient fibroblasts . In steady state ( 0 hours ) conditions , patient fibroblasts have a higher ratio of lipidated LC3 ( LC3II ) : LC3I than control cells . Induction of autophagy by nutrient starvation ( 2 hours incubation with minimal EBSS medium ) results in a raise in LC3II : LC3I ratio in both control and patient cells , but in VPS41S285P / R662 * fibroblasts this increase is only modest ( quantified in A ' ) ( n = 2 ) .

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

A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).


E . Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP / RFP tandem construct . The increased percentage of GFP / RFP - positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux ( quantified in E ' ) . > 12 Cells per cell line were quantified . Scale bars 10µm ; zoom , 1µm .

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E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.


A . Immunofluorescence of control , parental and patient fibroblasts labeled for LAMP - 1 and mTOR . In steady state conditions ( 0 hours ) VPS41S285P / R662 * fibroblasts show less colocalization between mTOR and LAMP - 1 . VPS41WT / WT , VPS41WT / S285P and VPS41WT / R662 * show an appropriate mTOR response upon nutrient deprivation ( 2 hours ) or restimulation ( 2 hours starvation followed by 15 minutes restimulation ) ( Appendix Fig S5A ) . > 10 Cells per cell line were quantified ( A ' ) . Quantifications are performed relative to colocalization under steady state conditions per cell line ( n = 3 ) . Scale bars , 10µm ; zoom , 1µm .

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

A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.


D . Rescue experiments of HeLaVPS41KO cells . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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

D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on cellular or secreted secretogranin II ( SgII ) of PC12 cells VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . Cells were washed and incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P .

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

B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( strain UA44 ) animals with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( strain UA391 ) fails to rescue neurodegeneration . At day 10 , none of the heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for the α - synuclein ( α - syn ) strain ( total of 90 worms ) and n = 90 for each independent transgenic strain ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative images of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows . Scale bar 20µm .

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

C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


( A ) Immunostaining of API in the vacuole of SEY6210 ( wild - type ) cell showing a dispersed pattern .

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(A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern.


( B - D ) Immunostaining of TVY1 ( pep4 ) cells showing API in the Cvt complex . The arrow points to a Cvt complex . Higher magnification images are shown in C and D .

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(B-D) Immunostaining of TVY1 (pep4) cells showing API in the Cvt complex. The arrow points to a Cvt complex. Higher magnification images are shown in C and D.


E ) Freeze - substitution fixation image of Cvt complex in TVY1 cell .

E) Freeze-substitution fixation image of Cvt complex in TVY1 cell.


( F ) Freeze - substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium .

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(F) Freeze-substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium.


( G ) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

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(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


( A and B ) Immunostaining image showing Cvt vesicles ( marked with arrows in A ) in the cytosol . Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle , respectively .

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(A and B) Immunostaining image showing Cvt vesicles (marked with arrows in A) in the cytosol. Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle, respectively.


( C ) Freeze - substitution fixation image of Cvt vesicles ( marked with arrows ) .

(C) Freeze-substitution fixation image of Cvt vesicles (marked with arrows).


( D ) Immunostaining of Cvt vesicle ( marked with arrows ) in the cytosol and Cvt body ( marked with double arrows ) in the vacuole .

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(D) Immunostaining of Cvt vesicle (marked with arrows) in the cytosol and Cvt body (marked with double arrows) in the vacuole.


( E and F ) Freeze - substitution fixation image of Cvt bodies in the vacuole ( marked with arrows ) . A Cvt vesicle is marked with an arrowhead .

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(E and F) Freeze-substitution fixation image of Cvt bodies in the vacuole (marked with arrows). A Cvt vesicle is marked with an arrowhead.


( G and H ) Freeze - substitution fixation image of Cvt vesicle contacting and fusing to a vacuole . V , Vacuole ; VM , vacuolar membrane .

O O O O O 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 and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Cvt complexes in TVY1 ( pep4 ) cells under nitrogen starvation conditions . Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium ( SD [ - N ] ) at 30 ° C for 30 min ( A and B ) or 60 min ( C and D ) and prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image showing a Cvt complex in an autophagosome ( marked by an arrow ) in the cytosol . ( B ) Freeze - substitution fixation image showing a Cvt complex in an autophagic body in the vacuole . ( C ) Immunostaining of a Cvt complex in an autophagic body in the vacuole . ( D ) Immunostaining of Cvt vesicles ( marked with arrows ) in the vacuole . AB , Autophagic body ; AP , autophagosome ; V , vacuole .

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Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.


Transport of Cvt complex in SEY6210 ( wild - type ) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions . Vegetative cells grown in SD medium were observed ( A - E ) . For starvation , logarithmically growing cells in SD were transferred to SD ( - N ) for 25 min ( F - H ) or 1 h ( I - K ) in the presence of 1 mM PMSF . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image of Cvt vesicle ( marked with an arrow ) . ( B ) Immunostaining of a Cvt vesicle ( marked with an arrow ) . ( C and D ) Higher magnification image of Cvt vesicle . Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle , respectively . ( E ) Freeze - substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex ( marked with an arrow ) . ( F ) Freeze - substitution image of the wrapping of a Cvt complex by the isolation membrane ( marked with an arrow ) . ( G ) Freeze - substitution image of a Cvt vesicle ( marked with an arrow ) fusing to a vacuole . ( H ) Freeze - substitution image of autophagic body containing a Cvt complex . ( I ) Immunostaining image depicting the wrapping of a Cvt complex . The arrow marks the enwrapping membrane . ( J ) Immunostaining of an autophagosome containing a Cvt complex . ( K ) Immunostaining of an autophagic body containing a Cvt complex in the vacuole . V , vacuole .

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Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.


Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes . ( A ) SEY6210 ( wild - type ) , cvt3 , and cvt7 yeast were grown in SD to 1 OD600 U / ml and transferred to SD ( - N ) . Aliquots were collected after 0 h ( + ) and 4 h ( − ) incubation and subjected to immunoblotting . The positions of precursor ( pro ) and mature ( m ) API are indicated . ( B ) cvt3 and cvt7 cells were pulse labeled for 10 min . After addition of cold cysteine and methionine , the cells were harvested , washed , and resuspended in either SD or SD ( - N ) and chased for the indicated times . API was recovered by immunoprecipitation , resolved on SDS polyacrylamide gels , and quantified using a phosphorimager ( STORM ; Molecular Dynamics , Sunnyvale , CA ) .

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

Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).


Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions . Vegetative cells were grown in SD medium at 30 ° C to log phase . For starvation , logarithmically growing cells in SD medium were transferred to SD ( - N ) for 5 h . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Immunostaining image depicting Cvt complex in the cytosol . Cvt vesicles are not detected in this strain . ( B ) Freeze - substitution fixation image showing Cvt complex in autophagosome ( marked with an arrow ) under starvation conditions , indicating that autophagy can still proceed . AP , Autophagosome ; N , nucleus ; V , vacuole .

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Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions. Vegetative cells were grown in SD medium at 30°C to log phase. For starvation, logarithmically growing cells in SD medium were transferred to SD(-N) for 5 h. Samples were prepared for electron microscopy as described in Fig. 1. (A) Immunostaining image depicting Cvt complex in the cytosol. Cvt vesicles are not detected in this strain. (B) Freeze-substitution fixation image showing Cvt complex in autophagosome (marked with an arrow) under starvation conditions, indicating that autophagy can still proceed. AP, Autophagosome; N, nucleus; V, vacuole.


A Heat map from RNA - Seq data from FACS - isolated muscle stem cells and single myofibers showing relative expression of Pax7 , Myf5 , MyoD , Myog and Myf6 .

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A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6.


H Colormap of Myf5 , MyoD and Myf6 peaks within 100kb of the Transcription Start Sites ( TSS ) of myokines ( supplemental table EV3 ) ranging from zero ( black ) to six peaks ( red ) occupancy . The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes .

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H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.


A Gene expression analysis of myokines ( table EV3 ) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media ( Ham ' s F10 supplemented with 20 % Fetal Bovine Serum , 1 % penicillin / streptomycin , 2 . 5 ng / ml basic Fibroblast Growth Factor ) to terminally differentiated myocytes ( 2 days in differentiation media , DMEM supplemented with 5 % horse serum ) to post mitotic multinucleated myotubes ( 5 days in differentiation media ) . Gene expression was assayed in biological triplicate by microarray ( Soleimani et al . , 2018 ; Soleimani et al . , 2012a ) . Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one . Expression values were then truncated at + / - 3 for color display . Blue indicates lower than average expression , while red indicates higher than average expression . B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ( table EV3 ) , similar to the analysis shown in ( A ) .

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A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).


C - E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP - Seq reads compared to control around EGF ( C ) , LIF and OSM ( D ) , and VEGFA ( E ) loci superimposed on reads for Histone H3 lysine 4 mono methyl ( H3K4me1 ) , Histone H3 lysine 4 tri methyl ( H3K4me3 ) and total H3 in myoblasts ( pink ) and myotubes ( green ) .

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C-E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (C), LIF and OSM (D), and VEGFA (E) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine 4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green).


F Snapshots of representative IGV tracks displaying RNA - Seq data from WT whole muscle ( WM ) , single myofibers ( SF ) and satellite cells ( SC ) showing the expression of EGF , EGFR and VEGFA . Ckm and Myf6 are used as positive controls for whole muscle and single fibers . PDGFRA , a marker of fibro / adipogenic progenitor cells , is used as a control to show the lack of niche cells in the single myofiber samples .

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F Snapshots of representative IGV tracks displaying RNA-Seq data from WT whole muscle (WM), single myofibers (SF) and satellite cells (SC) showing the expression of EGF, EGFR and VEGFA. Ckm and Myf6 are used as positive controls for whole muscle and single fibers. PDGFRA, a marker of fibro/adipogenic progenitor cells, is used as a control to show the lack of niche cells in the single myofiber samples.


G - H RNA - Sequencing Reads Per Million ( RPM ) for EGF ( G ) and EGFR ( H ) in satellite cells and single myofibers . RNA - Sequencing libraries were prepared from 1000 satellite cells freshly - isolated by FACS or from a single myofiber as described in the Material and Methods . ( n = 3 biological replicates per group ) bars = mean + / - SD , two - tailed t - test ) .

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G-H RNA-Sequencing Reads Per Million (RPM) for EGF (G) and EGFR (H) in satellite cells and single myofibers. RNA-Sequencing libraries were prepared from 1000 satellite cells freshly-isolated by FACS or from a single myofiber as described in the Material and Methods. (n=3 biological replicates per group) bars=mean +/- SD, two-tailed t-test).


I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control . Myofibers were cultured for 48 hours and were treated with 40ng / ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho - EGFR . Scale bar = 30μm .

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I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 40ng/ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho-EGFR. Scale bar=30μm.


J Activation of EGFR signaling in myoblasts treated with recombinant EGF . Myoblasts were treated with 2 ng / ml of recombinant protein in growth media ( Ham ' s F10 supplemented with 20 % FBS , 2 . 5 ng / ml bFGF ) for 15 minutes and lysed in RIPA lysis buffer . Western Blots were performed with antibodies against EGFR and phospho - EGFR ( pEGFR - Y1068 ) .

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J Activation of EGFR signaling in myoblasts treated with recombinant EGF. Myoblasts were treated with 2 ng/ml of recombinant protein in growth media (Ham's F10 supplemented with 20% FBS, 2.5 ng/ml bFGF) for 15 minutes and lysed in RIPA lysis buffer. Western Blots were performed with antibodies against EGFR and phospho-EGFR (pEGFR-Y1068).


K Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes ( 5DM ) . ( n = 3 biological replicates ) , two - tailed t - test , bars = mean + / - SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor ( EGF ) . ( n = 3 biological replicates ) , two - tailed t - test , bars = mean + / -

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K Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes (5DM). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor (EGF). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD


B Picture of 8 week - old Myf6 - KO and WT mice .

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B Picture of 8 week-old Myf6-KO and WT mice.


C Relative expression of Myf6 transcript in the hindlimb skeletal muscles of Myf6 - KO and WT mice measured by Quantitative Real - Time PCR ( RT - qPCR ) ( n = 4 mice ) . Boxes represent 1st and 3rd quartile , central line represents the median , whiskers represent the range , two - tailed t - test .

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C Relative expression of Myf6 transcript in the hindlimb skeletal muscles of Myf6-KO and WT mice measured by Quantitative Real-Time PCR (RT-qPCR) (n=4 mice). Boxes represent 1st and 3rd quartile, central line represents the median, whiskers represent the range, two-tailed t-test.


D Color map of genes that are up / down regulated in the whole muscle transcriptome ( RNA - Seq ) of injured and uninjured hindlimb muscles from Myf6 - KO and WT counterparts . Differential expression is Log2 transformed and truncated at + / - 3 . Color scale goes from blue ( - 3 = lower than mean ) , to yellow ( + 3 = higher than mean ) . E Heat map showing the distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ranging from zero ( black ) to six ChIP - Seq peaks ( red ) occupancy .

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D Color map of genes that are up/down regulated in the whole muscle transcriptome (RNA-Seq) of injured and uninjured hindlimb muscles from Myf6-KO and WT counterparts. Differential expression is Log2 transformed and truncated at +/-3. Color scale goes from blue (-3=lower than mean), to yellow (+3=higher than mean). E Heat map showing the distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ranging from zero (black) to six ChIP-Seq peaks (red) occupancy.


F Expression of EGF protein in the blood serum from Myf6 - KO and heterozygous counterparts as measured by Enzyme - Linked Immunosorbent Assay ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD G Expression of VEGFA protein in the blood serum from Myf6 - KO and heterozygous counterparts as measured by Enzyme - Linked Immunosorbent Assay ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD .

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F Expression of EGF protein in the blood serum from Myf6-KO and heterozygous counterparts as measured by Enzyme-Linked Immunosorbent Assay (ELISA). (n=5 heterozygous biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD G Expression of VEGFA protein in the blood serum from Myf6-KO and heterozygous counterparts as measured by Enzyme-Linked Immunosorbent Assay (ELISA). (n=5 heterozygous biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD.


H Expression of VEGFA protein in the secretome of Myf6 - KO and WT myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO and WT mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from the last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD I Expression of EGF protein in the secretome of Myf6 - KO and WT myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO and WT mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from the last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD J Expression of EGF protein in the secretome of Myf6 - HF expressing myotubes derived from Myf6 - KO myoblasts ( rescue ) compared to Myf6 - KO myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for five days . Media from the last 48 hours of differentiation ( days 3 to 5 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 3 Myf6 - KO technical replicates , n = 4 rescue technical replicates ) , two - tailed t - test , error bars = + / - SD .

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H Expression of VEGFA protein in the secretome of Myf6-KO and WT myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO and WT mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for seven days. Media from the last 48 hours of differentiation (days 5 to 7) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=4 WT biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD I Expression of EGF protein in the secretome of Myf6-KO and WT myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO and WT mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for seven days. Media from the last 48 hours of differentiation (days 5 to 7) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=4 WT biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD J Expression of EGF protein in the secretome of Myf6-HF expressing myotubes derived from Myf6-KO myoblasts (rescue) compared to Myf6-KO myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for five days. Media from the last 48 hours of differentiation (days 3 to 5) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=3 Myf6-KO technical replicates, n=4 rescue technical replicates), two-tailed t-test, error bars = +/- SD.


K RT - qPCR of Myf6 expression in Myf6 - KO ( n = 2 technical replicates ) , WT ( n = 3 biological replicates ) and Myf6 - HF ( rescue ) myotubes ( n = 3 technical replicates ) ( 5DM ) . " Rescue " myotubes were generated from Myf6 - KO myoblasts infected with a retrovirus containing Myf6 - HF . two - tailed t - test , error bars = + / -

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K RT-qPCR of Myf6 expression in Myf6-KO (n=2 technical replicates), WT (n=3 biological replicates) and Myf6-HF (rescue) myotubes (n=3 technical replicates) (5DM). "Rescue" myotubes were generated from Myf6-KO myoblasts infected with a retrovirus containing Myf6-HF. two-tailed t-test, error bars = +/- SD


L - M RT - qPCR of EGF ( L ) and VEGFA ( M ) expression in Myf6 - KO ( n = 5 ) and rescue ( n = 3 - 4 ) primary myotubes ( 5DM ) . two - tailed t - test , error bars = + / -

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L-M RT-qPCR of EGF (L) and VEGFA (M) expression in Myf6-KO (n=5) and rescue (n=3-4) primary myotubes (5DM). two-tailed t-test, error bars = +/- SD


Immunofluorescence staining of WT ( N ) and Myf6 - KO EDL ( O ) myofibers stained with PAX7 , EGFR , and phospho - EGFR antibodies . Live fibers were maintained in culture media for 44 hours , followed by 4 hours of serum starving in non - supplemented DMEM . For treated samples , 40ng / ml of recombinant EGF was added to the non - supplemented DMEM for 10 minutes before fixation . Activation status of EGFR was assessed using antibodies against phospho - EGFR ( pEGFR - Y1068 ) . Scale bar = 30μm P Quantification of the percentage of all pEGFR + / PAX7 + cells ( defined as PAX7 + cells showing low or high pEGFR signal ) on WT ( n = 24 fibers from n = 3 mice ) , Myf6 - KO ( n = 22 fibers from n = 3 mice ) and Myf6 - KO EDL myofibers treated with EGF ( n = 14 fibers from n = 3 mice ) . two - tailed t - test , error bars = + / - SD Q Quantification of the percentage of EGFRhigh / PAX7 + cells ( defined as PAX7 + cells showing high pEGFR signal ) on WT ( n = 37 fibers from n = 3 mice ) , Myf6 - KO ( n = 34 fibers from n = 3 mice ) and Myf6 - KO EDL myofibers treated with EGF ( n = 11 fibers from n = 3 mice ) . two - tailed t - test , error bars = + / -

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Immunofluorescence staining of WT (N) and Myf6-KO EDL (O) myofibers stained with PAX7, EGFR, and phospho-EGFR antibodies. Live fibers were maintained in culture media for 44 hours, followed by 4 hours of serum starving in non-supplemented DMEM. For treated samples, 40ng/ml of recombinant EGF was added to the non-supplemented DMEM for 10 minutes before fixation. Activation status of EGFR was assessed using antibodies against phospho-EGFR (pEGFR-Y1068). Scale bar=30μm P Quantification of the percentage of all pEGFR+/PAX7+ cells (defined as PAX7+ cells showing low or high pEGFR signal) on WT (n=24 fibers from n=3 mice) , Myf6-KO (n=22 fibers from n=3 mice) and Myf6-KO EDL myofibers treated with EGF (n=14 fibers from n=3 mice). two-tailed t-test, error bars = +/- SD Q Quantification of the percentage of EGFRhigh/PAX7+ cells (defined as PAX7+ cells showing high pEGFR signal) on WT (n=37 fibers from n=3 mice), Myf6-KO (n=34 fibers from n=3 mice) and Myf6-KO EDL myofibers treated with EGF (n=11 fibers from n=3 mice). two-tailed t-test, error bars = +/- SD


A Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 7 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm B Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 7 . ( n = 30 fields of view from n = 3 animals ) , two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 21 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm D Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 21 . ( n = 27 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD ) . E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 3 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm F Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 3 months of age . ( n = 52 fields of view from n = 5 animals ) , two tailed t - test , error bars = + / - SD . G Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 10 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm H Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 10 months of age . ( n = 60 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD .

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

A Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 7. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm B Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 7. (n=30 fields of view from n=3 animals), two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 21. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm D Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 21. (n=27 fields of view from n=3 animals) , two tailed t-test, error bars = +/- SD). E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 3 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm F Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 3 months of age. (n=52 fields of view from n=5 animals), two tailed t-test, error bars = +/- SD. G Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 10 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm H Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 10 months of age. (n=60 fields of view from n=3 animals), two tailed t-test, error bars = +/- SD.


Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 3 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm J Quantification of the number of MuSCs per fiber between 3 month - old Myf6 - KO ( n = 41 fibers from n = 3 mice ) and WT ( n = 31 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / - SD K Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 10 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm L Quantification of the number of MuSCs per fiber between 10 month - old Myf6 - KO ( n = 38 fibers from n = 2 mice ) and WT ( n = 67 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / -

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

Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm J Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO (n=41 fibers from n=3 mice) and WT (n=31 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD K Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm L Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO (n=38 fibers from n=2 mice) and WT (n=67 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD


Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7 . Scale bar = 100μm , arrowheads highlight select MuSC B Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at postnatal day 7 ( n = 15 fields of view from n = 3 animals ) Two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21 . Scale bar = 100μm , arrowheads highlight select MuSC D Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO ( n = 28 fields of view from n = 3 animals ) and WT mice ( n = 31 fields of view from n = 3 animals ) at postnatal day 21 . Two - tailed t - test , error bars = + / - SD E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at 3 months of age . Scale bar = 100μm , arrowheads highlight select MuSC F Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at 3 months of age ( n = 30 fields of view from n = 5 mice ) . Two - tailed t - test , error bars = + / -

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

Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7. Scale bar=100μm, arrowheads highlight select MuSC B Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at postnatal day 7 (n=15 fields of view from n=3 animals) Two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21. Scale bar=100μm, arrowheads highlight select MuSC D Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO (n=28 fields of view from n=3 animals) and WT mice (n=31 fields of view from n=3 animals) at postnatal day 21. Two-tailed t-test, error bars = +/- SD E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at 3 months of age. Scale bar=100μm, arrowheads highlight select MuSC F Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at 3 months of age (n=30 fields of view from n=5 mice). Two-tailed t-test, error bars = +/- SD


H ( Left ) Heat Map of up and down - regulated genes in the p38 MAPK pathway ( WP400 ) in Myf6 - KO and WT satellite cells . ( Right ) Gene set enrichment analysis of p38 MAPK pathway genes . In each plot , the blue curve represents the gene set enrichment score profile ( Subramanian et al . , 2005 ) across p38 MAPK pathway genes that are ranked by their differential expression . The grey curve represents the null expectation . P - values are based on permutation test ( 10 , 000 permutations ) .

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

H (Left) Heat Map of up and down-regulated genes in the p38 MAPK pathway (WP400) in Myf6-KO and WT satellite cells. (Right) Gene set enrichment analysis of p38 MAPK pathway genes. In each plot, the blue curve represents the gene set enrichment score profile (Subramanian et al., 2005) across p38 MAPK pathway genes that are ranked by their differential expression. The grey curve represents the null expectation. P-values are based on permutation test (10,000 permutations).


Immunofluorescent analysis of WT and Myf6 - KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI . Scale bar = 200μm J Quantification of the fusion index between Myf6 - KO ( n = 54 fields of view from n = 3 mice ) and WT ( n = 81 fields of view from n = 4 mice ) 7DM myotubes . Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view . Two - tailed t - test , error bars = + / -

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

Immunofluorescent analysis of WT and Myf6-KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI. Scale bar=200μm J Quantification of the fusion index between Myf6-KO (n=54 fields of view from n=3 mice) and WT (n=81 fields of view from n=4 mice) 7DM myotubes. Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view. Two-tailed t-test, error bars = +/- SD


EDL myofibers isolated from WT and Myf6 - KO mice fixed at time T = 0 and stained with PAX7 , MYOD and DAPI . Scale bar = 30μm L Quantification of the percentage of MYOD + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 88 myofibers ) vs WT ( n = 86 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD M Quantification of the average percentage of MYOD + / PAX7 + satellite cells per mouse in Myf6 - KO and WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

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

EDL myofibers isolated from WT and Myf6-KO mice fixed at time T=0 and stained with PAX7, MYOD and DAPI. Scale bar=30μm L Quantification of the percentage of MYOD+/PAX7+ satellite cells per fiber in Myf6-KO (n= 88 myofibers) vs WT (n= 86 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD M Quantification of the average percentage of MYOD+/PAX7+ satellite cells per mouse in Myf6-KO and WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Staining for phospho - p38 MAPK on EDL myofibers cultured for 48 hours from Myf6 - KO and WT mice . Scale bar = 30μm O Quantification of the percentage of phospho - p38 + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 56 myofibers ) vs WT ( n = 67 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD P Quantification of the average percentage of phospho - p38 + / PAX7 + satellite cells per mouse in Myf6 - KO vs WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

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

Staining for phospho-p38 MAPK on EDL myofibers cultured for 48 hours from Myf6-KO and WT mice. Scale bar=30μm O Quantification of the percentage of phospho-p38+/PAX7+ satellite cells per fiber in Myf6-KO (n=56 myofibers) vs WT (n=67 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD P Quantification of the average percentage of phospho-p38+/PAX7+ satellite cells per mouse in Myf6-KO vs WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Western blot analysis of C2C12 myoblasts treated with EGF , LIF , or EGF + LIF while in differentiation media and stained with phosphor - p38 , total p38 , myogenin , MF20 and GAPDH . Cells were treated with EGF ( 50ng / ml ) , LIF ( 1000U / ml ) or EGF + LIF after 48 hours in differentiation media , and every 6 hours following initial treatment , followed by cell lysis at 72 hours . R Phospho - p38 western blot signal intensity normalized to total p38 signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / -

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

Western blot analysis of C2C12 myoblasts treated with EGF, LIF, or EGF+LIF while in differentiation media and stained with phosphor-p38, total p38, myogenin, MF20 and GAPDH. Cells were treated with EGF (50ng/ml), LIF (1000U/ml) or EGF+LIF after 48 hours in differentiation media, and every 6 hours following initial treatment, followed by cell lysis at 72 hours. R Phospho-p38 western blot signal intensity normalized to total p38 signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD


EDL myofibers treated repeatedly with recombinant EGF protein ( 100ng / ml ) at 0h , 24h , 32h , 40h , and 48h in culture , and fixed at 48 hours , compared to untreated controls . V Quantification of the number of PAX7 + cells per fiber in EGF - treated ( n = 19 fibers from n = 1 mouse ) and untreated control EDL myofibers ( n = 16 fibers from n = 1 mouse ) after 48 hours in culture . Two - tailed t - test , error bars = + / -

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EDL myofibers treated repeatedly with recombinant EGF protein (100ng/ml) at 0h, 24h, 32h, 40h, and 48h in culture, and fixed at 48 hours, compared to untreated controls. V Quantification of the number of PAX7+ cells per fiber in EGF-treated (n=19 fibers from n=1 mouse) and untreated control EDL myofibers (n=16 fibers from n=1 mouse) after 48 hours in culture. Two-tailed t-test, error bars = +/- SD


B Representative images of bioluminescent signal from hindlimbs of Myf6 - KO and WT counterparts at day 1 , 7 , 14 , and 21 post transplantation . Scale bar =

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

B Representative images of bioluminescent signal from hindlimbs of Myf6-KO and WT counterparts at day 1, 7, 14, and 21 post transplantation. Scale bar=1cm


C Time course of average bioluminescent signal ( total flux in radiance ) in Myf6 - KO and WT mice at 1 , 7 , 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs ( mean across 4 - 5 animals per group ± SEM at each time point , two - tailed t - test ) .

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C Time course of average bioluminescent signal (total flux in radiance) in Myf6-KO and WT mice at 1, 7, 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs (mean across 4-5 animals per group ± SEM at each time point, two-tailed t-test).


D Bioluminescent signal ( radiance ) in Myf6 - KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation . ( n = 4 - 5 animals per group ± SD ) two - tailed t - test , bars represent mean + / - SD .

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D Bioluminescent signal (radiance) in Myf6-KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation. (n=4-5 animals per group ± SD) two-tailed t-test, bars represent mean +/- SD.


E - F Immunostaining of TA cross - sections of injured muscle from 3 month - old WT ( E ) and Myf6 - KO ( F ) mice stained with laminin ( LAMA1 ) and PAX7 antibodies . Acute muscle injury was performed by injection of Cardiotoxin ( CTX ) into the TA muscle of Myf6 - KO and WT mice ( n = 5 animals per group ) . Arrowheads indicate PAX7 + MuSCs . Scale bar = 100μm G Quantification of the number of PAX7 + cells per unit area of TA muscle cross sections between Myf6 - KO and WT mice ( n = 5 animals per group ) two - tailed t - test , bars represent mean + / -

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

E-F Immunostaining of TA cross-sections of injured muscle from 3 month-old WT (E) and Myf6-KO (F) mice stained with laminin (LAMA1) and PAX7 antibodies. Acute muscle injury was performed by injection of Cardiotoxin (CTX) into the TA muscle of Myf6-KO and WT mice (n=5 animals per group). Arrowheads indicate PAX7+ MuSCs. Scale bar=100μm G Quantification of the number of PAX7+ cells per unit area of TA muscle cross sections between Myf6-KO and WT mice (n= 5 animals per group) two-tailed t-test, bars represent mean +/- SD


H Immunofluorescent analysis of Myf6 - KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies . Scale bar = 20μm IQuantification of the number of PAX7 + MuSCs per fiber in Myf6 - KO ( n = 27 fibers from n = 3 mice ) and WT ( n = 29 fibers from n = 3 mice ) mice after 48 hours in culture . Two - tailed t - test , error bars = + / - SD .

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

H Immunofluorescent analysis of Myf6-KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies. Scale bar=20μm IQuantification of the number of PAX7+ MuSCs per fiber in Myf6-KO (n=27 fibers from n=3 mice) and WT (n=29 fibers from n=3 mice) mice after 48 hours in culture. Two-tailed t-test, error bars = +/- SD.