Figure 7 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 -

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

Figure 7 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-test


Figure 8Wild 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 - testWild 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 - 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Wild 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-testWild 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-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 immunofluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test


Figure 2A . 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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Figure 2A. 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.


Figure 3D . 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 .

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Figure 3D. 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.


Figure 4A . 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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Figure 4A. 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.


Figure 5A . 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 .

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Figure 5A. 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.


Figure 6B . 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 .

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Figure 6B. 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.


Figure 7C . 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 .

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Figure 7C. 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.


Figure 1 ( 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 . 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 . ( G ) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

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Figure 1(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.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.(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


Figure 2 ( 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 ) . ( 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 . ( G and H ) Freeze - substitution fixation image of Cvt vesicle contacting and fusing to a vacuole . V , Vacuole ; VM , vacuolar membrane .

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Figure 2(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).(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.(G and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Figure 3Cvt 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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Figure 3Cvt 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.


Figure 4Transport 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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Figure 4Transport 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.


Figure 5Mutant 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 ) .

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Figure 5Mutant 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).


Figure 6Analysis 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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Figure 6Analysis 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.


Figure 1A 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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Figure 1A 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.


Figure 2A 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 ) . 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 ) . 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 ) . 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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Figure 2A 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).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).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).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


Figure 3B 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 . 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 . 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 = + / - SDL - 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 = + / - SDImmunofluorescence 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 = + / -

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B 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.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.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 = +/- SDL-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 = +/- SDImmunofluorescence 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


Figure 4A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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 = +/- SD


Figure 5Immunofluorescent 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 = + / - SDH ( 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 = + / - SDEDL 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 = + / - SDStaining 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 = + / - SDWestern 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 = + / - SDEDL 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 = + / -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunofluorescent 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 = +/- SDH (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 = +/- SDEDL 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 = +/- SDStaining 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 = +/- SDWestern 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 = +/- SDEDL 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


Figure 6B Representative images of bioluminescent signal from hindlimbs of Myf6 - KO and WT counterparts at day 1 , 7 , 14 , and 21 post transplantation . Scale bar = 1cmC 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 . 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 + / - SDH 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 . J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF ( 1000U / ml ) or EGF ( 80ng / ml ) every 8 hours . Fibers were stained with antibodies against PAX7 and MyoD . Scale bar = 30μm K Quantification of the number of PAX7 + and MYOD + cells on the EDL myofiber treated with recombinant LIF ( n = 80 fibers ) , EGF ( n = 50 fibers ) or vehicle control ( n = 61 fibers ) . Two - tailed t - test , bars represent mean + / - SDEdU staining of cultured primary myoblasts isolated by FACS ( see materials and methods ) from Myf6 - KO and WT mice . Staining was analyzed after 6 , 12 and 24 hours of EdU incorporation . Scale bar = 100μm M Quantification of the percentage of EdU + myoblasts after 6 hours ( WT n = 75 fields of view from n = 3 mice , Myf6 - KO n = 72 fields of view from n = 3 mice ) , 12 hours ( WT n = 82 fields of view from n = 3 mice , Myf6 - KOn = 82 fields of view from n = 3 mice ) , and 24 hours ( WT n = 70 fields of view from n = 3 mice , Myf6 - KO n = 65 fields of view from n = 3 mice ) of EdU incorporation . Scale bar = 100μmRepresentative images of a co - culture assay between WT myotubes and either WT or Myf6 - KO myoblasts . Briefly , WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6 - KO myoblasts for an additional 2 days , followed by immunostaining for MF20 and DAPI . Scale bar = 400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes , for 2 additional days ( n = 3 biological replicates ) . Two - tailed t - test , bars represent mean + / - SD .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B Representative images of bioluminescent signal from hindlimbs of Myf6-KO and WT counterparts at day 1, 7, 14, and 21 post transplantation. Scale bar=1cmC 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.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 +/- SDH 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.J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF (1000U/ml) or EGF (80ng/ml) every 8 hours. Fibers were stained with antibodies against PAX7 and MyoD. Scale bar=30μm K Quantification of the number of PAX7+ and MYOD+ cells on the EDL myofiber treated with recombinant LIF (n=80 fibers), EGF (n=50 fibers) or vehicle control (n=61 fibers). Two-tailed t-test, bars represent mean +/- SDEdU staining of cultured primary myoblasts isolated by FACS (see materials and methods) from Myf6-KO and WT mice. Staining was analyzed after 6, 12 and 24 hours of EdU incorporation. Scale bar=100μm M Quantification of the percentage of EdU+ myoblasts after 6 hours (WT n=75 fields of view from n=3 mice, Myf6-KO n=72 fields of view from n=3 mice), 12 hours (WT n=82 fields of view from n=3 mice, Myf6-KOn=82 fields of view from n=3 mice), and 24 hours (WT n=70 fields of view from n=3 mice, Myf6-KO n=65 fields of view from n=3 mice) of EdU incorporation. Scale bar=100μmRepresentative images of a co-culture assay between WT myotubes and either WT or Myf6-KO myoblasts. Briefly, WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6-KO myoblasts for an additional 2 days, followed by immunostaining for MF20 and DAPI. Scale bar=400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes, for 2 additional days (n=3 biological replicates). Two-tailed t-test, bars represent mean +/- SD.


Figure 1B OsPRR73 confers salinity stress tolerance in rice . The seedlings of Dongjin ( DJ , the wild type control ) , osprr73 , and the complementation line of osprr73 ( abbreviated as Com - L1 ) grown under 12 h light / 12 h dark conditions for 28 days ( left panel ) , transferred to 180 mM NaCl for 21 days ( middle panel ) and recovered for 12 and 24 days ( the two right panels ) respectively . Scale bar , 5 cm . C Statistical analysis of the survival rate of DJ , osprr73 and Com - L1 plants in ( B ) after recovery 12 days . Data are presented as mean ± SD . ( n from 4 biological replicates , and 24 plants were tested in each of biological replicates ) . ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test . E Null mutant of osprr73 in Nipponbare ( NIP ) background by genome editing displayed hypersensitivity to NaCl treatment . Scale bar , 5 cm . F Survival rate of NIP ( wild type of CRISPR lines ) and osprr73 - C ( C stands for CRISPR ) plants in ( E ) after recovery 9 days . Data are presented as mean ± SD . n = 4 biological replicates , 24 plants for each biological replicate . ( * ) P ≤ 0 . 05 and ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test . Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 . Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days . Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B OsPRR73 confers salinity stress tolerance in rice. The seedlings of Dongjin (DJ, the wild type control), osprr73, and the complementation line of osprr73 (abbreviated as Com-L1) grown under 12 h light/12 h dark conditions for 28 days (left panel), transferred to 180 mM NaCl for 21 days (middle panel) and recovered for 12 and 24 days (the two right panels) respectively. Scale bar, 5 cm. C Statistical analysis of the survival rate of DJ, osprr73 and Com-L1 plants in (B) after recovery 12 days. Data are presented as mean ± SD. (n from 4 biological replicates, and 24 plants were tested in each of biological replicates). (***) P ≤ 0.001 were generated by student's t-test.E Null mutant of osprr73 in Nipponbare (NIP) background by genome editing displayed hypersensitivity to NaCl treatment. Scale bar, 5 cm. F Survival rate of NIP (wild type of CRISPR lines) and osprr73-C (C stands for CRISPR) plants in (E) after recovery 9 days. Data are presented as mean ± SD. n = 4 biological replicates, 24 plants for each biological replicate. (*) P ≤0.05 and (***) P ≤ 0.001 were generated by student's t-test.Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days. Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.


Figure 3A , B The Na + and K + contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days . Na + ( A ) and K + ( B ) contents were measured with ICP method . Data represent means ± SD , ( n = 3 , biological replicates ) . ( * * ) P ≤ 0 . 001 and ( * ) P ≤ 0 . 001 indicate significant difference by student t - test . The abbreviation of n . s . stands for no significant . C Ratio of Na + to K + was calculated with their respective content in ( A ) and ( B ) . Data represent mean ± SD ( n = 3 , biological replicates ) . For each of biological replicates , 6 individual plants were measured . The asterisk represents significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , and n . s . indicates no significant . D Four - weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d ( middle panel ) and recovered for additional 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rates with Na2SO4 treatment in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants for each biological replicate . ( * * * ) P ≤ 0 . 001 indicates significant difference by student t - test . F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . G Statistical analysis of survival rates with MgCl2 treatment in ( F ) . The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days . Data represent means ± SD . n = 2 biological replicates , 24 plants of each replicate . H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . I The survival rate of DJ and osprr73 plants in mannitol stress . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . The abbreviation of n . s . stands for no significant generated by student t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B The Na+ and K+ contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days. Na+ (A) and K+ (B) contents were measured with ICP method. Data represent means ± SD, (n = 3, biological replicates). (**) P ≤ 0.001 and (*) P ≤ 0.001 indicate significant difference by student t-test. The abbreviation of n.s. stands for no significant. C Ratio of Na+ to K+ was calculated with their respective content in (A) and (B). Data represent mean ± SD (n = 3, biological replicates). For each of biological replicates, 6 individual plants were measured. The asterisk represents significant difference among means by student's t-test with (*) P ≤0.05, and n.s. indicates no significant.D Four-weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d (middle panel) and recovered for additional 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rates with Na2SO4 treatment in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants for each biological replicate. (***) P ≤ 0.001 indicates significant difference by student t-test. F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. G Statistical analysis of survival rates with MgCl2 treatment in (F). The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days. Data represent means ± SD. n = 2 biological replicates, 24 plants of each replicate. H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. I The survival rate of DJ and osprr73 plants in mannitol stress. Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. The abbreviation of n.s. stands for no significant generated by student t-test.


Figure 4A , B The expression level of OsHKT2 ; 1 and OsHKT1 ; 5 in osprr73 mutant and the wild control Dongjin ( DJ ) plants with or without salinity stress in multiple time points . DJ Data represent means ± SD . n = 3 , technical replicates . The representative data is from at least two individual biological replicates . The asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 and ( * * * ) P ≤ 0 . 001 . C Transient expression assay in the infiltrated leaves of N . benthamiana showing OsPRR73 can repress OsHKT2 ; 1 transcription . Left panel showing the bioluminescence signal of OsHKT2 ; 1 and OsPRR73 . Bioluminescence of co - transformation of either GFP or 35S : GFP - OsPRR73 ( GFP - OsPRR73 ) with OsHKT2 ; 1pro : LUC in N . benthamiana . D Quantiﬁcation of bioluminescence intensity of OsHKT2 ; 1pro : LUC shown in ( C ) . Data represent means ± SD . n = 10 . The asterisks indicate the significant difference by student ' s t - test with ( * * * ) P ≤ 0 . 001 . At least two biological replicates were conducted with similar results . E ChIP - qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2 ; 1 promoter by OsPRR73 , compared to wild type DJ controls . The amplicon of Ubiquitin was taken as a negative control . Two - week - old seedlings were harvested at ZT12 . Data represent means ± SD ( n = 3 , technical repeats ) . The experiments were performed at least two biological replicates with similar result . Top scheme indicates the locations of amplicons for ChIP analysis . The asterisks represent significant difference among means by student ' s t - test with ( * * ) P ≤ 0 . 01 . F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2 ; 1 promoter . Unlabelled probes were used as competitor with indicated folds . MBP alone was used as negative control . Arrowhead marks the shifted positive bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B The expression level of OsHKT2;1 and OsHKT1;5 in osprr73 mutant and the wild control Dongjin (DJ) plants with or without salinity stress in multiple time points. DJ Data represent means ± SD. n = 3, technical replicates. The representative data is from at least two individual biological replicates. The asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01 and (***) P ≤ 0.001.C Transient expression assay in the infiltrated leaves of N. benthamiana showing OsPRR73 can repress OsHKT2;1 transcription. Left panel showing the bioluminescence signal of OsHKT2;1 and OsPRR73. Bioluminescence of co-transformation of either GFP or 35S: GFP-OsPRR73 (GFP-OsPRR73) with OsHKT2;1pro: LUC in N. benthamiana. D Quantiﬁcation of bioluminescence intensity of OsHKT2;1pro: LUC shown in (C). Data represent means ± SD. n = 10. The asterisks indicate the significant difference by student's t-test with (***) P ≤ 0.001. At least two biological replicates were conducted with similar results.E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01.F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2;1 promoter. Unlabelled probes were used as competitor with indicated folds. MBP alone was used as negative control. Arrowhead marks the shifted positive bands.


Figure 5A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo . CoIP was conducted with GFP - Trap beads , and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated . B Split luciferase complementation ( SLC ) assay showing OsPRR73 interacts with HDAC10 in the co - infiltrated N . benthamiana leaves . C Bimolecular - fluorescence complementation ( BiFC ) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus . BF represents bright field . Scale bar , 40 µm . D N - terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10 . Upper panel shows the schematic illustration of full - length and the truncated OsPRR73 proteins . CoIP was performed with GFP - Trap beads , and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively . E , F Chromatin Immunoprecipitation - qPCR ( ChIP - qPCR ) assay to detect the enriched promoter regions of OsHKT2 ; 1 by using the H3K9ac ( E ) and H3 ( F ) antibodies . Two - week - old seedlings were harvested at ZT12 . The locations of amplicons were described in Figure 4E . Data represent means ± SD . n = 3 technical replicates . The asterisk indicates the significant difference by student ' s t - test with ( * ) P ≤ 0 . 05 . The experiments were repeated at least twice with similar result .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo. CoIP was conducted with GFP-Trap beads, and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated.B Split luciferase complementation (SLC) assay showing OsPRR73 interacts with HDAC10 in the co-infiltrated N.benthamiana leaves.C Bimolecular-fluorescence complementation (BiFC) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus. BF represents bright field. Scale bar, 40 µm.D N-terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10. Upper panel shows the schematic illustration of full-length and the truncated OsPRR73 proteins. CoIP was performed with GFP-Trap beads, and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively.E, F Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay to detect the enriched promoter regions of OsHKT2;1 by using the H3K9ac (E) and H3 (F) antibodies. Two-week-old seedlings were harvested at ZT12. The locations of amplicons were described in Figure 4E. Data represent means ± SD. n = 3 technical replicates. The asterisk indicates the significant difference by student's t-test with (*) P ≤ 0.05. The experiments were repeated at least twice with similar result.


Figure 6D The oshkt2 ; 1 seedling is more tolerant to the treatment of NaCl . The oshkt2 ; 1 and NIP , its wild type , four - week - old seedlings grown under NaCl conditions for 0 days ( left panel ) , then were transferred to 200 mM NaCl for 28 days ( middle panel ) , and recovered for 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rate in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . ( * ) P ≤ 0 . 05 was generated by student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D The oshkt2;1 seedling is more tolerant to the treatment of NaCl. The oshkt2;1 and NIP, its wild type, four-week-old seedlings grown under NaCl conditions for 0 days (left panel), then were transferred to 200 mM NaCl for 28 days (middle panel), and recovered for 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rate in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. (*) P ≤ 0.05 was generated by student's t-test.