Figure 4E Geranylgeranylation activity of WT GGTase - III and the indicated mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) and 3H - GGPP ( 1 μM ) were used as substrates ( mean ± SEM , n = 3 ) . F Kinetic analysis of WT GGTase - III and the site 1 or site 2 mutant ( 100 nM each ) using increasing concentrations of Cys195 - farnesyl Ykt6ΔAIM ( mean ± SEM , n = 3 for WT and Y306A , n = 1 for V48A / V50A ) . Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase - III . WT and mutant Ykt6 proteins ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) .

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Figure 4E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A).Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


Figure 5H Geranylgeranylation activity of WT GGTase - III and tunnel mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) and 3H - GGPP ( 1 μM ) were used as substrates ( mean ± SEM , n = 3 ) . I GGPP binding to GGTase - III . WT GGTase - III and tunnel mutants ( 1 μM each ) were incubated with 3H - GGPP ( 2 μM ) at 4ºC for 10 min . After desalting , enzyme - bound 3H - GGPP was quantified by scintillation counting ( mean ± SEM , n = 3 ) .

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Figure 5H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3).


Figure 6A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti - Ykt6 antibody . Note the slight difference in the gel mobility of Ykt6 ( arrows ) . B Prenylation status of rat brain Ykt6 . Recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , Cys194 / 195 - diprenyl Ykt6 samples ( 1 ng each ) , and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate ( DOC ) - containing buffer and analyzed by immunoblotting with anti - Ykt6 antibody ( DOC - PAGE ; upper panel ) . The same samples were analyzed by conventional SDS - PAGE and immunoblotting with anti - Ykt6 antibody ( SDS - PAGE ; lower panel ) . C Prenylation status of Ykt6 in WT and PTAR1 KO cells . Recombinant Ykt6 samples and cell lysates of WT cells , PTAR1 KO cells , and PTAR1 KO cells stably expressing PTAR1 ( KO + PTAR1 ) were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , and analyzed by immunoblotting with anti - Ykt6 antibody . D In vitro reconstitution of Ykt6 double prenylation . Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase - III ( 100 nM ) or RabGGTase ( 100 nM ) in the absence or presence of GGPP ( 10 μM ) . After incubation , reaction products were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , and analyzed by immunoblotting with anti - Ykt6 antibody .

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 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 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 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

Figure 6A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti-Ykt6 antibody. Note the slight difference in the gel mobility of Ykt6 (arrows).B Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, Cys194/195-diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)-containing buffer and analyzed by immunoblotting with anti-Ykt6 antibody (DOC-PAGE; upper panel). The same samples were analyzed by conventional SDS-PAGE and immunoblotting with anti-Ykt6 antibody (SDS-PAGE; lower panel).C Prenylation status of Ykt6 in WT and PTAR1 KO cells. Recombinant Ykt6 samples and cell lysates of WT cells, PTAR1 KO cells, and PTAR1 KO cells stably expressing PTAR1 (KO + PTAR1) were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.D In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase-III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.


Figure 7A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells . Cells were co - immunostained for GM130 ( a cis - Golgi marker ) and golgin - 97 ( a trans - Golgi marker ) . Images were deconvoluted using Huygens software . The lower panels show magnified images of the boxed region of the upper panels . Scale bars , 5 μm . B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells . Arrows indicate Golgi stacks . Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells . Scale bars , 500 nm . Defect in intra - Golgi trafficking in PTAR1 KO cells . ( C ) VSVG - GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC . VSVG - GFP fluorescence images taken at the indicated times after the temperature shift are shown . The right panel shows quantification of VSVG - GFP fluorescence in the Golgi region after the temperature shift ( mean ± SEM , n = 6 ) . Defect in intra - Golgi trafficking in PTAR1 KO cells . ( D ) VSVG - GFP expressing WT HeLa cells , PTAR1 KO HeLa cells , and PTAR1 KO HeLa cells stably expressing PTAR1 ( KO + PTAR1 ) were cultured at 40ºC and then shifted to 32ºC . At the indicated time points , cell surface proteins were biotinylated using sulfo - NHS - LC - biotin . Biotinylated VSVG - GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti - GFP antibody . The right panel shows quantification of the cell surface biotinylated VSVG - GFP ( means ± SEM , n = 3 ) . Data were analyzed by one - way ANOVA with Dunnett ' s post - hoc test . * p < 0 . 05 , * * * p < 0 . 001 . E Analysis of LAMP1 glycosylation . Cell lysates of WT HeLa cells , PTAR1 KO HeLa cells , and PTAR1 KO HeLa cells stably expressing PTAR1 ( KO + PTAR1 ) were analyzed by immunoblotting with anti - LAMP1 antibody ( upper ) . Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin ( MAL ) agarose and analyzed by immunoblotting ( lower ) . F Defect in the Golgi SNARE assembly in PTAR1 KO cells . The Golgi SNARE complex was immunoprecipitated from NEM - treated WT HAP1 cells , PTAR1 KO HAP1 cells , and PTAR1 KO HAP1 cells stably expressing PTAR1 ( KO + PTAR1 ) using control mouse IgG , anti - GS28 IgG , or anti - syntaxin 5 IgG . Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5 , GS28 , GS15 , and Ykt6 . Syntaxin 5 has two isoforms with different translation initiation sites . Inputs were 20 % ( syntaxin 5 , GS28 , and GS15 ) and 2 % ( Ykt6 ) . The data shown are representative of three independent experiments with similar results .

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

Figure 7A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells. Cells were co-immunostained for GM130 (a cis-Golgi marker) and golgin-97 (a trans-Golgi marker). Images were deconvoluted using Huygens software. The lower panels show magnified images of the boxed region of the upper panels. Scale bars, 5 μm.B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells. Arrows indicate Golgi stacks. Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells. Scale bars, 500 nm.Defect in intra-Golgi trafficking in PTAR1 KO cells. (C) VSVG-GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC. VSVG-GFP fluorescence images taken at the indicated times after the temperature shift are shown. The right panel shows quantification of VSVG-GFP fluorescence in the Golgi region after the temperature shift (mean ± SEM, n = 6).Defect in intra-Golgi trafficking in PTAR1 KO cells. (D) VSVG-GFP expressing WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were cultured at 40ºC and then shifted to 32ºC. At the indicated time points, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin. Biotinylated VSVG-GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti-GFP antibody. The right panel shows quantification of the cell surface biotinylated VSVG-GFP (means ± SEM, n = 3). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, ***p < 0.001.E Analysis of LAMP1 glycosylation. Cell lysates of WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were analyzed by immunoblotting with anti-LAMP1 antibody (upper). Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin (MAL) agarose and analyzed by immunoblotting (lower).F Defect in the Golgi SNARE assembly in PTAR1 KO cells. The Golgi SNARE complex was immunoprecipitated from NEM-treated WT HAP1 cells, PTAR1 KO HAP1 cells, and PTAR1 KO HAP1 cells stably expressing PTAR1 (KO + PTAR1) using control mouse IgG, anti-GS28 IgG, or anti-syntaxin 5 IgG. Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5, GS28, GS15, and Ykt6. Syntaxin 5 has two isoforms with different translation initiation sites. Inputs were 20% (syntaxin 5, GS28, and GS15) and 2% (Ykt6). The data shown are representative of three independent experiments with similar results.


Figure 1 ( B - C ) Immunofluorescent ( IF ) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors . Scale bar = 100 µm . ( D ) Representative images of Ctrl , Heterozygous ( Het ) and YY1cKO newborn ( P0 ) mice or embryos at E18 . 5 day . ( E ) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test . The lung from YY1cKO pup sank whereas Ctrl floated . ( F ) P0 lungs were stained with Hematoxylin and Eosin ( H & E ) and representative images are shown . Scale bar = 100 µm . ( G ) Representative images of diaphragms ( Dps ) isolated from P0 Ctrl or YY1cKO littermates . ( H ) The above Dps were used for H & E or Immunohistochemistry ( IHC ) staining for MyHC . Representative images from 3 pairs of littermates are shown . Scale bar = 50 µm . The above harvested Dps were subjected to IF staining for MyHC together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested Dps were subjected to IF staining for Troponin T ( TnT ) together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) . ( K ) Representative images of H & E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18 . 5 day . Scale bar = 400 µm ( left ) or 50 µm ( right ) . The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of MyHC + fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested limb muscles were subjected to IF staining for TnT together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of TnT + fibers per area are shown on the right ( n = 3 mice , each ) . The above harvested limb ( N ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 100 µm ( N ) Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . The above harvested diaphragm ( O ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 50 µm ( O ) . Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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 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 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

Figure 1(B-C) Immunofluorescent (IF) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors. Scale bar = 100 µm.(D) Representative images of Ctrl, Heterozygous (Het) and YY1cKO newborn (P0) mice or embryos at E18.5 day.(E) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test. The lung from YY1cKO pup sank whereas Ctrl floated.(F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates.(H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm.The above harvested Dps were subjected to IF staining for MyHC together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).The above harvested Dps were subjected to IF staining for Troponin T (TnT) together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).(K) Representative images of H&E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18.5 day. Scale bar = 400 µm (left) or 50 µm (right).The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of MyHC+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb muscles were subjected to IF staining for TnT together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of TnT+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb (N) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 100 µm (N) Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.The above harvested diaphragm (O) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 50 µm (O). Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01 ***P<0.001.


Figure 2 ( B ) SCs were FACS sorted 3 days after the last TM injection and cultured for 1 . 5 days ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells . ( C ) IF staining for Pax7 and YY1 on freshly isolated FISCs or ( D ) single myofibers from EDL muscles or ( E ) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells . Scale bar = 100 µm in ( C ) or 50 µm in ( D - E ) . ( F ) Representative FACS plots . About 100 , 000 cells from 2 - month - old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post - TM injection . The percentage of SCs was shown . ( n = 3 mice , each ) . ( G ) H & E staining was performed on the injured TA muscles collected from the designated times post - CTX injection to visualize the degree of regeneration . Scale bar = 100 µm . ( H ) IF staining for eMyHC on the TA muscles 5 days post - CTX injury . Quantifications of the number of eMyHC + fibers are shown on the right ( n = 3 , each ) . Scale bar = 50 µm . ( I ) Left : representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post - CTX injury . Right : Masson ' s Trichrome staining of the above muscles to visualize the degree of fibrosis . Scale bar = 100 µm . ( J ) Cross - section area ( CSA ) of individual myofibers in TA muscles 4 weeks after injury was measured . The distribution for CSA was calculated ( n = 3 mice ) . ( K ) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice . Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) . Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of MyoD + SCs per area are shown on the right ( n = 3 mice , each ) . ( N ) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice . Quantifications of the numbers of Pax7 + SCs per 100 fibers are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . ( P ) H & E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury . Scale bar = 400 μm ( left ) or 100 μm ( right ) . ( Q ) Representative images of above TA muscles 7 days after second injury . ( R ) Quantifications of the weight above TA muscles from three pairs of littermate mice . ( S ) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice . Quantifications of the number of eMyHC + fibers per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . ( T ) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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 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 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 B-PANEL_START 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 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 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

Figure 2(B) SCs were FACS sorted 3 days after the last TM injection and cultured for 1.5 days; RT-qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells.(C) IF staining for Pax7 and YY1 on freshly isolated FISCs or (D) single myofibers from EDL muscles or (E) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells. Scale bar =100 µm in (C) or 50 µm in (D-E).(F) Representative FACS plots. About 100,000 cells from 2-month-old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post-TM injection. The percentage of SCs was shown. (n=3 mice, each).(G) H&E staining was performed on the injured TA muscles collected from the designated times post-CTX injection to visualize the degree of regeneration. Scale bar = 100 µm.(H) IF staining for eMyHC on the TA muscles 5 days post-CTX injury. Quantifications of the number of eMyHC+ fibers are shown on the right (n=3, each). Scale bar = 50 µm.(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.(J) Cross-section area (CSA) of individual myofibers in TA muscles 4 weeks after injury was measured. The distribution for CSA was calculated (n=3 mice).(K) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice.Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of Pax7+ SCs per area are shown on the right (n=3 mice, each).Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of MyoD+ SCs per area are shown on the right (n=3 mice, each).(N) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). Scale bar = 100 μm.(P) H&E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury. Scale bar = 400 μm (left) or 100 μm (right).(Q) Representative images of above TA muscles 7 days after second injury.(R) Quantifications of the weight above TA muscles from three pairs of littermate mice.(S) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice. Quantifications of the number of eMyHC+ fibers per area are shown on the right (n=3 mice, each). Scale bar = 100 μm.(T) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7+ SCs per area are shown on the right (n=3 mice, each). Scale bar = 100 μm. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


Figure 3 ( B ) SCs were FACS sorted 4 months after the last TM injection ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells . ( C ) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration . ( D ) Representative images of limb muscles ( Tibialis anterior , TA ; Gastrocnemius , Gas ; Quadruple , Quad ) from Ctrl or YY1dKO mice . ( E ) Quantifications of muscle weight from three pairs of littermate mice . ( F ) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection . ( G ) H & E or ( H ) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice . Quantifications of the numbers of MyHC + fibers per area of TA or Dp muscles are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm . ( I ) Collagen1 or ( J ) Masson ' s Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice . Scale bar = 100 µm . ( K ) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6 - month - old Ctrl and YY1dKO mice was calculated ( n = 3 mice , each ) . ( L ) Representative FACS plots showing the percentage of SCs sorted from 6 - month - old Ctrl and YY1dKO mice ( n = 3 mice , each ) . ( M ) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice . Quantifications of the numbers of Pax7 + SCs per 100 fibers are shown on the right ( n = 3 mice , each ) . EDL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force . Left : Representative trace images of normalized tetanic force . ( Right ) : quantification of the force by normalizing with cross - sectional area ( CSA ) . ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force . Left : Representative trace images of normalized tetanic force . ( Right ) : quantification of the force by normalizing with cross - sectional area ( CSA ) . ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-PANEL_START 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 B-PANEL_START 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 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 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 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 O O O O

Figure 3(B) SCs were FACS sorted 4 months after the last TM injection; RT-qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells.(C) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration.(D) Representative images of limb muscles (Tibialis anterior, TA; Gastrocnemius, Gas; Quadruple, Quad) from Ctrl or YY1dKO mice. (E) Quantifications of muscle weight from three pairs of littermate mice.(F) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection.(G) H&E or (H) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Quantifications of the numbers of MyHC+ fibers per area of TA or Dp muscles are shown on the right (n=3 mice, each). Scale bar = 100 µm.(I) Collagen1 or (J) Masson's Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Scale bar = 100 µm.(K) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6-month-old Ctrl and YY1dKO mice was calculated (n=3 mice, each).(L) Representative FACS plots showing the percentage of SCs sorted from 6-month-old Ctrl and YY1dKO mice (n=3 mice, each).(M) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each).EDL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


Figure 4 ( A ) FACS - isolated SCs from Pax7 - nGFP mice were cultured for the designated time ( 0 , 24 , 48 or 72 hrs ) . YY1 expression was quantified by RT - qPCR . 18S was used as the normalization control . ( B ) Flow cytometric analysis of YY1 protein or IgG control in FACS - isolated SCs cultured for the designated time ( 0 , 24 or 48hrs ) . YY1 protein level is normalized to IgG levels . The numbers indicate the normalized level of YY1 protein relative to that of FISCs . ( C ) An equal number of FACS - isolated SCs from Ctrl or YY1iKO mice were cultured for 48 hrs and EdU labelled for 8 hrs , followed by immunostaining for Pax7 ( green ) and EdU ( red ) . Quantifications of the percentage of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm . ( D ) The above cells were also IF stained for Pax7 ( red ) and MyoD ( green ) . Quantifications of each population , Pax7 + MyoD + , or Pax7 - MyoD + , are shown on the right ( n = 3 , each ) . Scale bar = 100 µm . ( E ) Single EDL myofibers were cultured for 48 hrs before staining for Pax7 ( red ) and MyoD ( green ) . Quantifications of the numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on the right ( n = 3 mice , each ) . Scale bar = 50 µm . ( F ) Pax7 ( green ) and Ki67 ( red ) staining was performed on the above single EDL myofibers . Scale bar = 50 µm . Quantifications of the numbers of each population , Pax7 + Ki67 - , Pax7 - Ki67 + , or Pax7 + Ki67 + , are shown on the right ( n = 3 mice , each ) . ( G ) SCs expansion in vivo was determined by EdU labeling for 12 hrs 2 . 5 days after CTX injury . Quantifications of the numbers of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm . ( H ) An equal number of FACS - isolated SCs from Ctrl or YY1iKO mice were cultured for 24hrs and EdU labeled for 12 hrs , followed by immunostaining for Pax7 ( green ) and EdU ( red ) was performed . Quantifications of the percentage of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm . Single EDL myofibers were cultured for 12 ( I ) hrs in growth media before staining for Pax7 ( green ) and MyoD ( red ) . Scale bar = 50 µm . Quantifications of the proportion of MyoD + cells are shown on the right ( n = 3 mice , each ) . Single EDL myofibers were cultured for 24 ( J ) hrs in growth media before staining for Pax7 ( green ) and MyoD ( red ) . Scale bar = 50 µm . Quantifications of the proportion of MyoD + cells are shown on the right ( n = 3 mice , each ) . ( K ) FACS - isolated SCs from YY1iKO mice were infected with YY1 expressing or control viruses , followed by immunostaining for Pax7 ( green ) and MyoD ( red ) 36 hrs post - transfection . Scale bar = 100 µm . Quantifications of the numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on the right ( n = 3 mice , each ) . ( L ) Single EDL myofibers were cultured for 3 days , followed by immunostaining for Myogenin ( red ) and MyoD ( green ) . Scale bar = 50 µm . Quantifications of the numbers of each population , MyoD + Myog + , MyoD - Myog + , or MyoD + Myog - , are shown on the right ( n = 3 mice , each ) . ( M ) FACS - isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium ; the degree of differentiation was assessed by IF staining of MF20 + . Scale bar = 100 µm . Quantifications of the numbers of MF20 + area per nucleus are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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 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 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

Figure 4(A) FACS-isolated SCs from Pax7-nGFP mice were cultured for the designated time (0, 24, 48 or 72 hrs). YY1 expression was quantified by RT-qPCR. 18S was used as the normalization control.(B) Flow cytometric analysis of YY1 protein or IgG control in FACS-isolated SCs cultured for the designated time (0, 24 or 48hrs). YY1 protein level is normalized to IgG levels. The numbers indicate the normalized level of YY1 protein relative to that of FISCs.(C) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 48 hrs and EdU labelled for 8 hrs, followed by immunostaining for Pax7 (green) and EdU (red). Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.(D) The above cells were also IF stained for Pax7 (red) and MyoD (green). Quantifications of each population, Pax7+MyoD+, or Pax7-MyoD+, are shown on the right (n=3, each). Scale bar = 100 µm.(E) Single EDL myofibers were cultured for 48 hrs before staining for Pax7 (red) and MyoD (green). Quantifications of the numbers of each population, Pax7+MyoD-, Pax7-MyoD+, or Pax7+MyoD+, are shown on the right (n=3 mice, each). Scale bar = 50 µm.(F) Pax7 (green) and Ki67 (red) staining was performed on the above single EDL myofibers. Scale bar = 50 µm. Quantifications of the numbers of each population, Pax7+Ki67-, Pax7-Ki67+, or Pax7+Ki67+, are shown on the right (n= 3 mice, each).(G) SCs expansion in vivo was determined by EdU labeling for 12 hrs 2.5 days after CTX injury. Quantifications of the numbers of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.(H) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 24hrs and EdU labeled for 12 hrs, followed by immunostaining for Pax7 (green) and EdU (red) was performed. Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.Single EDL myofibers were cultured for 12 (I) hrs in growth media before staining for Pax7 (green) and MyoD (red). Scale bar = 50 µm. Quantifications of the proportion of MyoD+ cells are shown on the right (n=3 mice, each).Single EDL myofibers were cultured for 24 (J) hrs in growth media before staining for Pax7 (green) and MyoD (red). Scale bar = 50 µm. Quantifications of the proportion of MyoD+ cells are shown on the right (n=3 mice, each).(K) FACS-isolated SCs from YY1iKO mice were infected with YY1 expressing or control viruses, followed by immunostaining for Pax7 (green) and MyoD (red) 36 hrs post-transfection. Scale bar = 100 µm. Quantifications of the numbers of each population, Pax7+MyoD-, Pax7-MyoD+, or Pax7+MyoD+, are shown on the right (n=3 mice, each).(L) Single EDL myofibers were cultured for 3 days, followed by immunostaining for Myogenin (red) and MyoD (green). Scale bar = 50 µm. Quantifications of the numbers of each population, MyoD+Myog+, MyoD-Myog+, or MyoD+Myog-, are shown on the right (n=3 mice, each).(M) FACS-isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium; the degree of differentiation was assessed by IF staining of MF20+. Scale bar = 100 µm. Quantifications of the numbers of MF20+ area per nucleus are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


Figure 5 ( A ) RNA - seq was performed with RNAs extracted from ASCs ( FACS purified and cultured for 36hrs ) of YY1iKO or Ctrl mice ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl . ( C ) Genomic snapshots showing the examples of mitochondrial genes ( Uqcr11 , Mrp11 , Apo5o and Nme4 ) up - regulated in YY1iKO vs Ctrl cells . ( D ) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells ( p - value < 0 . 0001 ) . ( E ) RT - qPCR validation of the expression of the selected up - regulated mitochondrial genes in YY1iKO vs Ctrl . ( F ) Protein levels of the respiratory chain complexes I - V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail . β - actin was used as the loading control . ( G ) RNA - seq was performed using RNAs from freshly isolated SCs ( FISCs ) ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl . ( I ) Genomic snapshots show the selected mitochondrial genes ( Cox8b , Nme4 , Slc25a29 , Nnt ) up - regulated in YY1iKO vs Ctrl FISCs . ( J ) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown . ( N ) Genomic snapshots of two of the above identified mitochondrial genes ( Tomm40I and Ndufa13 ) that are bound by YY1 in their TSS ( ChIPseq tracks ) and up - regulated by YY1 deletion ( RNAseq tracks ) . ( O ) ChIP - qPCR validation of YY1 binding on some mitochondrial genes . Negative control ( NC ) represents a genomic region on Chromosome 11 with no YY1 binding peak identified . Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control . ChIP - qPCR was performed to show the enrichment of Ezh2 ( P ) binding on selected mitochondrial genes . ChIP - qPCR was performed to show the enrichment of Suz12 ( Q ) binding on selected mitochondrial genes . ChIP - qPCR was performed to show the enrichment of H3K27 me3 ( R ) binding on selected mitochondrial genes . ( S ) A construct to express wild type YY1 ( WT ) , or two mutants lacking DNA binding activity , YY1 ( 1 - 394 ) and YY1 ( S365D ) were transfected into YY1iKO cells ; the expression of mitochondrial gene was measured . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 B-PANEL_START 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 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 5(A) RNA-seq was performed with RNAs extracted from ASCs (FACS purified and cultured for 36hrs) of YY1iKO or Ctrl mice; scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.(C) Genomic snapshots showing the examples of mitochondrial genes (Uqcr11, Mrp11, Apo5o and Nme4) up-regulated in YY1iKO vs Ctrl cells.(D) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells (p-value < 0.0001).(E) RT-qPCR validation of the expression of the selected up-regulated mitochondrial genes in YY1iKO vs Ctrl.(F) Protein levels of the respiratory chain complexes I-V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail. β-actin was used as the loading control.(G) RNA-seq was performed using RNAs from freshly isolated SCs (FISCs); scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.(I) Genomic snapshots show the selected mitochondrial genes (Cox8b, Nme4, Slc25a29, Nnt) up-regulated in YY1iKO vs Ctrl FISCs.(J) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown.(N) Genomic snapshots of two of the above identified mitochondrial genes (Tomm40I and Ndufa13) that are bound by YY1 in their TSS (ChIPseq tracks) and up-regulated by YY1 deletion (RNAseq tracks).(O) ChIP-qPCR validation of YY1 binding on some mitochondrial genes. Negative control (NC) represents a genomic region on Chromosome 11 with no YY1 binding peak identified. Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control.ChIP-qPCR was performed to show the enrichment of Ezh2 (P) binding on selected mitochondrial genes.ChIP-qPCR was performed to show the enrichment of Suz12 (Q) binding on selected mitochondrial genes.ChIP-qPCR was performed to show the enrichment of H3K27 me3 (R) binding on selected mitochondrial genes.(S) A construct to express wild type YY1(WT), or two mutants lacking DNA binding activity, YY1(1-394) and YY1(S365D) were transfected into YY1iKO cells; the expression of mitochondrial gene was measured. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


Figure 6 ( A ) An equal number of FACS - isolated SCs from C57 mice were cultured for 24 hrs and treated with 0 , 1 , 10 , or 20mM 2 - DG ( glycolytic inhibitor ) or NaN3 ( respiration inhibitor ) for 3 hrs before measurement of ATP production . The relative ATP levels normalized to the 0 mM values are plotted . N = 3 mice . ( B ) SCs from C57 mice were treated with 10mM 2 - DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay ( n = 3 mice , each ) . ( C ) SCs from C57 mice were treated with 10mM 2 - DG for 24 hrs and subject to EdU labeling for 6 hrs . The percentage of Pax7 + / EdU + cells over the total number of Pax7 + cells was quantified ( n = 3 mice , each ) . ( D ) Ctrl and YY1iKO ASCs were stained with 60 ug / mL 2 - NBDG for 45 mins and the fluorescence intensity ( MFI ) of 2 - NBDG was measured by flow cytometry ( n = 3 mice , each ) . An equal number of Ctrl and YY1iKO cells were cultured for 36h ; basal extracellular acidification rate ( ECAR ) level were measured and normalized with cell numbers . N = 3 mice . An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration ( F ) were measured and normalized with cell numbers . N = 3 mice . byAn equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production ( G ) were measured and normalized with cell numbers . N = 3 mice . ( H ) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes ( Hk2 , Glut1 , Ldha , Pdk1 , Eno1 ) down - regulated in YY1iKO vs Ctrl ASCs . ( I ) Expression of the selected glycolytic genes was quantified by RT - qPCR . Hsp90ab1 was used as the normalization control . ( J ) Re - expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up - regulation of glycolytic genes including Glut1 , Pfkl , Gapdh , Eno1 , Pgam1 , Pkm . Hsp90ab1 was used as the normalization control . ( K ) FACS - isolated SCs from Pax7 - nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs . The percentage of MyoD + EdU + cells over the total number of MyoD + cells was quantified . Scale bar = 100 µm . N = 3 mice . ( L ) The expression of glycolytic genes and cell cycle related genes was detected by RT - qPCR in the above transfected cells . β - actin was used as the normalization control . ( M ) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs . β - actin was used as the loading control . ( N ) Left : Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs . Right : Relative mean fluorescence intensity ( MFI ) of Hif1α protein is shown . N = 3 mice . ( O ) YY1iKO ASCs were transfected with empty YY1 WT viruses ; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N = 3 mice . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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 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 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 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 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 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 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

Figure 6(A) An equal number of FACS-isolated SCs from C57 mice were cultured for 24 hrs and treated with 0, 1, 10, or 20mM 2-DG (glycolytic inhibitor) or NaN3 (respiration inhibitor) for 3 hrs before measurement of ATP production. The relative ATP levels normalized to the 0 mM values are plotted. N=3 mice.(B) SCs from C57 mice were treated with 10mM 2-DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay (n=3 mice, each).(C) SCs from C57 mice were treated with 10mM 2-DG for 24 hrs and subject to EdU labeling for 6 hrs. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified (n=3 mice, each).(D) Ctrl and YY1iKO ASCs were stained with 60 ug/mL 2-NBDG for 45 mins and the fluorescence intensity (MFI) of 2-NBDG was measured by flow cytometry (n=3 mice, each).An equal number of Ctrl and YY1iKO cells were cultured for 36h; basal extracellular acidification rate (ECAR) level were measured and normalized with cell numbers. N=3 mice.An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration (F) were measured and normalized with cell numbers. N=3 mice. byAn equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production (G) were measured and normalized with cell numbers. N=3 mice.(H) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes (Hk2, Glut1, Ldha, Pdk1, Eno1) down-regulated in YY1iKO vs Ctrl ASCs.(I) Expression of the selected glycolytic genes was quantified by RT-qPCR. Hsp90ab1 was used as the normalization control.(J) Re-expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up-regulation of glycolytic genes including Glut1, Pfkl, Gapdh, Eno1, Pgam1, Pkm. Hsp90ab1 was used as the normalization control.(K) FACS-isolated SCs from Pax7-nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.(L) The expression of glycolytic genes and cell cycle related genes was detected by RT-qPCR in the above transfected cells. β-actin was used as the normalization control.(M) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs. β-actin was used as the loading control.(N) Left: Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs. Right: Relative mean fluorescence intensity (MFI) of Hif1α protein is shown. N=3 mice.(O) YY1iKO ASCs were transfected with empty YY1 WT viruses; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N=3 mice. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


Figure 7 ( A - B ) Ctrl or YY1iKO ASCs were treated with CHX ( Cycloheximide ) for 90 mins and Hif1α protein was measured by flow cytometry . The numbers indicate the degradation rate after CHX treatment . N = 3 mice . ( C ) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO ( 11 . 14 % ± 1 . 71 % ) vs Ctrl ( 5 . 08 % ± 0 . 796 % ) . N = 3 mice . ( D ) Lysates from C2C12 myoblasts ( GM ) were subjected to co - immunoprecipitation ( Co - IP ) assays with anti - YY1 or anti - IgG antibodies and blotted with anti - Hif1α or anti - YY1 antibodies . ( E ) The above lysates were also IPed with anti - Hif1α or anti - IgG antibodies and blotted with anti - YY1 or anti - Hif1α antibodies . ( F ) ASCs from YY1iKO mice were infected with pRlenti - Hif1α ( P402A / P564A ) ( Hif1α - DM ) viruses to overexpress a non - degradable Hif1α protein or GFP - expressing viruses . ASCs from Ctrl mice were also infected with the GFP viruses . 36 hrs after infection , the expression of target glycolytic genes was detected by RT - qPCR . Hsp90ab1 was used as the normalization control . ( G ) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses . EdU labeling was then performed for 5 . 5 hrs . The percentage of MyoD + EdU + cells over the total number of MyoD + cells was quantified . Scale bar = 100 µm . N = 3 mice . Heat maps indicating gene expression levels ( Log2 [ FPKM ] ) of pentose cycle enzymes ( H ) in YY1iKO vs Ctrl . Heat maps indicating gene expression levels ( Log2 [ FPKM ] ) of amino acid transporter ( I ) in YY1iKO vs Ctrl . ( J ) YY1 ( WT ) or YY1 ( S365D ) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs . ( K ) Similarly , YY1 ( WT ) or YY1 ( 1 - 394 ) mutant was expressed and EdU labeled for 6hrs . A GFP expressing plasmid was used the negative control . The percentage of Pax7 + / EdU + cells over the total number of Pax7 + cells was quantified . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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 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 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 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

Figure 7(A-B) Ctrl or YY1iKO ASCs were treated with CHX (Cycloheximide) for 90 mins and Hif1α protein was measured by flow cytometry. The numbers indicate the degradation rate after CHX treatment. N=3 mice. (C) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO (11.14% ± 1.71%) vs Ctrl (5.08% ± 0.796%). N=3 mice.(D) Lysates from C2C12 myoblasts (GM) were subjected to co-immunoprecipitation (Co-IP) assays with anti-YY1 or anti-IgG antibodies and blotted with anti-Hif1α or anti-YY1 antibodies.(E) The above lysates were also IPed with anti-Hif1α or anti-IgG antibodies and blotted with anti-YY1 or anti-Hif1α antibodies.(F) ASCs from YY1iKO mice were infected with pRlenti-Hif1α (P402A/P564A) (Hif1α-DM) viruses to overexpress a non-degradable Hif1α protein or GFP-expressing viruses. ASCs from Ctrl mice were also infected with the GFP viruses. 36 hrs after infection, the expression of target glycolytic genes was detected by RT-qPCR. Hsp90ab1 was used as the normalization control.(G) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses. EdU labeling was then performed for 5.5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.Heat maps indicating gene expression levels (Log2[FPKM]) of pentose cycle enzymes (H) in YY1iKO vs Ctrl.Heat maps indicating gene expression levels (Log2[FPKM]) of amino acid transporter (I) in YY1iKO vs Ctrl.(J) YY1(WT) or YY1(S365D) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs. (K) Similarly, YY1 (WT) or YY1 (1-394) mutant was expressed and EdU labeled for 6hrs. A GFP expressing plasmid was used the negative control. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


Figure 1A . Results of CRISPR screening analyzed by the MAGeCK algorithm . B . Wild type ( WT ) and NCOA4 KO MEFs were pretreated with 25 μg / ml ferric ammonium citrate ( FAC ) for 16 h and then chased with 20 μg / ml cycloheximide ( CHX ) for the indicated times . Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies . C . WT MEFs untreated or treated with 10 μg / ml FAC for 12 h were lysed with SDS - containing sample buffer or Triton buffer , and analyzed by immunoblotting with the indicated antibodies . E . Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12 h , or 100 μM Deferoxamine ( Dfo ) for 6 h after treatment with 10 μg / ml FAC for 12 h were prepared by differential centrifugation as described in ( D ) . Each fraction was subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies . F . WT MEFs were untreated , treated with 10 μg / ml FAC for 12h or treated with 10 μg / ml FAC , 10 μM E64d and 10 μg / ml pepstatin A ( pep A ) for 12h . The cells were immunostained with anti - NCOA4 and anti - LAMP1 . Scale bar , 10 μm . Quantitative data are shown as the means ± SD of three biological replicates . At least 30 cells were quantified in each replicate . P = 0 . 033 ( Welch Two Sample t - test ) . G . NCOA4 puncta were photobleached in MEFs stably expressing NCOA4 - GFP after treatment with 10 μg / ml FAC for 12 h , and then fluorescent recovery was monitored . p62 puncta were photobleached in MEFs stably expressing GFP - human p62 , and then fluorescent recovery was monitored . Representative images are shown . Time 0 indicates the start of recovery after photobleaching . Scale bars , 2 μm . H . Quantitative data of fluorescence recovery in ( G ) shown as means ± SD . 26 dots ( NCOA4 ) or 25 dots ( p62 ) were quantified from three biological replicates . I . Time - lapse imaging of MEFs stably expressing NCOA4 - GFP . Representative fusion and fission images are shown . Red arrowheads indicate the fission event . Scale bars , 5 μm .

O B-PANEL_START 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 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 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 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 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

Figure 1A. Results of CRISPR screening analyzed by the MAGeCK algorithm.B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.


Figure 2B . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h and then fixed for imaging . Cells were immunostained with anti - myc antibody . Representative images are shown . Scale bar , 10 μm . C . Number of puncta per cell in ( B ) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates . D . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h , fractionated , and analyzed by immunoblotting with the indicated antibodies . E . DISOPRED3 disorder score of human NCOA4 . F . WT MEFs stably expressing NCOA4 IDR ( aa 167 - 334 ) fused with tandem FKBP were treated with 10 μg / ml FAC or 20 μM Dfo for 12 h and then cultured with 0 . 1 nM FKBP ligand ( AP20187 ) for 6h . The cells were fixed for imaging and immunostained with anti - myc to analyze puncta of the fusion protein . Scale bar , 10μm . G . Number of puncta in ( F ) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates . P < 0 . 0001 ( Dfo + ligand vs FAC + ligand ) and P = 0 . 048 ( FAC vs FAC + ligand ) ( Kruskal - Wallis ANOVA with Dunn ' s multiple comparison test ) . P values were adjusted using the Bonferroni method . H . Coomassie - stained SDS - PAGE gel of purified proteins . I . The amount of co - purifying iron in purified proteins in ( H ) was measured by inductively coupled plasma mass spectrometry ( ICP - MS ) . Data are shown as means ± SD of three biological replicates . P = 0 . 032 ( Welch Two Sample t - test ) .

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Figure 2B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.E. DISOPRED3 disorder score of human NCOA4.F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).


Figure 3A . Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated . Samples were subjected to SDS - PAGE and stained with Coomassie Brilliant Blue . B . Quantitative results in ( A ) are shown as means ± SEM of three biological replicates . F . Electron micrograph of NCOA4 KO MEFs reconstituted with myc - hNCOA4 . Cryosections were labeled with anti - myc antibody . Scale bar , 200nm . G . Quantification of the diameter of NCOA4 condensates . Red line shows the mean . The dot plot represents the diameter of 36 condensates from two biological replicates .

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Figure 3A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm. G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.


Figure 4A . MEFs were treated with 10 μg / ml FAC for the indicated times and harvested . Cell lysates were fractionated and analyzed by SDS - PAGE with the indicated antibodies . Asterisks indicate a non - specific band . TfR1 ; Transferrin receptor 1B . MEFs were cultured for 12 h in medium containing 10 μg / ml FAC supplemented with DMSO or 10 μM E64d and 10 μg / ml pepstatin A ( pepA ) . Samples were analyzed by immunoblotting with the indicated antibodies . The black arrowhead indicates an intact ferritin band , and the white arrowhead indicates a partial degradation band of ferritin . C . NCOA4 KO MEFs reconstituted with hNCOA4 - myc were cultured with 10 μg / ml FAC for the indicated times and fixed for imaging . Cells were immunostained with ferritin and myc antibodies . Scale bar , 10μm . D . Myc ( NCOA4 ) puncta were extracted , and average signal intensities of myc and ferritin in the puncta in ( C ) were plotted . E . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min . Cells were lysed with Triton buffer , and lysates were pulled down by streptavidin beads . Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies . F . WT MEFs pretreated with 10 μg / ml FAC for 6 h or 24 h were chased with 20 μg / ml CHX and analyzed by immunoblotting with the indicated antibodies . G . NCOA4 KO MEFs reconstituted with myc - hNCOA4 were pretreated with 10 μg / ml FAC and then cultured with 20 μg / ml CHX or 200 nM bafilomycin A1 ( BafA ) . Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies . Scale bar , 10μm .

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Figure 4A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.


Figure 5A . WT , ATG7 KO , TAX1BP1 KO , and FIP200KO MEFs were treated or not treated with 10 μg / ml FAC for 12 h . Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies . C . HEK293T cells were transfected with GFP - NCOA4 and the indicated FLAG - His - TAX1BP1 variants . Soluble lysates extracted from transfected cells were immunoprecipitated with anti - FLAG antibody . Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies . WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg / ml FAC for 12 h . ( D , F ) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies . WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg / ml FAC for 12 h . ( E ) Cells were fixed for imaging and immunostained to analyze ferritin localization . Scale bar , 10μm .

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Figure 5A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml FAC for 12 h. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.


Figure 6A . MEFs were pretreated with 10 μg / ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time . Cells were lysed with SDS - containing sample buffer or Triton buffer . Lysates were analyzed by immunoblotting with the indicated antibodies . B . MEFs were treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12h , or 100 μM Dfo for 6 h after treatment with 10 μg / ml FAC for 12 h . Triton - soluble lysates were subjected to glycerol gradient ultracentrifugation analysis . C . WT and TAX1BP1 KO MEFs were pretreated with 25 μg / ml FAC for 16 h and then cultured with 100 μM Dfo . Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies . D . Soluble FTL bands in ( C ) were quantified by densitometry . Data are shown as the means ± SD of three biological replicates . E . TAX1BP1 KO MEFs ( sg1 clone ) stably expressing FLAG - OsTIR1 ( F74G ) and FLAG - mAID - mTAX1BP1 were pretreated with 10 μg / ml FAC for 12 h , cultured with DMSO or 1 μM 5 - Ph - IAA for 2 h , and then treated with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with the indicated antibodies . 5 - Ph - IAA , a derivative of Auxin ; mAID , mini auxin - inducible degronF . TAX1BP1 KO MEFs ( clone sg1 ) in which ATG7 was knocked down were pretreated with 10 μg / ml FAC for 12 h and then cultured with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with the indicated antibodies . G . TAX1BP1 KO MEFs ( clone sg1 ) stably expressing myc - hNCOA4 pretreated with 25 μg / ml FAC for 16 h were cultured with 100 μM Dfo , and soluble lysates were analyzed by immunoblotting with the indicated antibodies . H , I . NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg / ml FAC for 12 h and then chased with ( H ) 20 μg / ml CHX or ( I ) 100 μM Dfo for the indicated times . Soluble lysates were analyzed by immunoblotting with the indicated antibodies . J . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 3 h ( indicated as F ) or 100 μM DFO for 3 h after treatment with 10 μg / ml FAC for 12 h ( indicated as D ) , and then cultured with DMSO or 50 μM biotin for 30 min . Triton - soluble cell lysates were pulled down by streptavidin beads . Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies . The black and white arrowheads indicate hNCOA4 - TurboID and endogenous NCOA4 , respectively .

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Figure 6A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degronF. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.


Figure 1A IMR90 ER : RasV12 primary fibroblasts were cultured in the presence of EtOH ( proliferating ) or 100nM 4OHT ( for 6 - 8days to induce RasV12 expression and oncogene - induced senescence , OIS ) . mRNA levels of 4OHT cells , normalised to PUM1 and presented relative to EtOH control . ( n = 4 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) B , C Cells as in A were analysed by Western blot for Lamp1 , p62 and Cathepsin B and quantified , normalised to GAPDH and expressed relative to EtOH . ( n = 3 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001D , E Cells as in A were fixed and immunostained for Lamp1 and signal intensity was quantified . Scale bars : 10µm ( n = 3 independent experimental repeats ( at least 40 cells analysed per condition , from at least 4 fields of view , per repeat ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) K , Cells as in A were incubated with DQ - BSA and TR - BSA for 2 hours , fixed and imaged by confocal microscopy . Scale bar : 20µm ( n = 3 independent experimental repeats ( at least 240 cells analysed from 8 fields of view per condition ) . O , Cells as in A were incubated with 50µg / ml cycloheximide ( CHX ) for the timepoints indicated . Degradation of autophagy receptors , p62 and NDP52 was assessed by Western blot ( n = 3 independent experimental repeats ) .

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Figure 1A IMR90 ER:RasV12 primary fibroblasts were cultured in the presence of EtOH (proliferating) or 100nM 4OHT (for 6-8days to induce RasV12 expression and oncogene-induced senescence, OIS). mRNA levels of 4OHT cells, normalised to PUM1 and presented relative to EtOH control. (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)B, C Cells as in A were analysed by Western blot for Lamp1, p62 and Cathepsin B and quantified, normalised to GAPDH and expressed relative to EtOH. (n=3 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001D, E Cells as in A were fixed and immunostained for Lamp1 and signal intensity was quantified. Scale bars: 10µm (n=3 independent experimental repeats (at least 40 cells analysed per condition, from at least 4 fields of view, per repeat). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)K, Cells as in A were incubated with DQ-BSA and TR-BSA for 2 hours, fixed and imaged by confocal microscopy. Scale bar: 20µm (n=3 independent experimental repeats (at least 240 cells analysed from 8 fields of view per condition).O, Cells as in A were incubated with 50µg/ml cycloheximide (CHX) for the timepoints indicated. Degradation of autophagy receptors, p62 and NDP52 was assessed by Western blot (n=3 independent experimental repeats).


Figure 2A Representative images of GFP - TFEB localisation in EtOH and 4OHT - treated fibroblasts , in full nutrient conditions . Scale bar : 20µm . B , EtOH and 4OHT - treated fibroblasts immunostained for endogenous TFE3 in the conditions indicated ; starvation indicates serum starvation overnight and one - hour amino acid starvation , refeeding was with full nutrient medium ( including FCS ) for 2 hours . Cells were incubated with leptomycin B ( LMB ) for 3 hours and where indicated , wash out involved replacement of the media for a further 2 hours . Scale bar : 20µm ; ( n = 3 independent experimental repeats ( at least 150 cells analysed from at least 5 fields of view per repeat ) . C EtOH and 4OHT - treated fibroblasts in the conditions indicated ; starvation indicates serum starvation overnight and one - hour amino acid starvation , refeeding was with full nutrient medium ( including FCS ) for 2 hours . Cells were incubated with leptomycin B ( LMB ) for 3 hours and where indicated , wash out involved replacement of the media for a further 2 hours . % cells with nuclear TFE3 was quantified . ( n = 3 independent experimental repeats ( at least 150 cells analysed from at least 5 fields of view per repeat ) . Data information : Violin blots include all datapoints from n = 3 , lines represent median and upper and lower quartiles ; Analysis : one - way ANOVA with Tukey ' s multiple comparison test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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Figure 2A Representative images of GFP-TFEB localisation in EtOH and 4OHT-treated fibroblasts, in full nutrient conditions. Scale bar: 20µm.B, EtOH and 4OHT-treated fibroblasts immunostained for endogenous TFE3 in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. Scale bar: 20µm; (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat).C EtOH and 4OHT-treated fibroblasts in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. % cells with nuclear TFE3 was quantified. (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat). Data information: Violin blots include all datapoints from n=3, lines represent median and upper and lower quartiles; Analysis: one-way ANOVA with Tukey's multiple comparison test * p<0.05; ** p<0.01; *** p<0.001.


Figure 3D Cells treated as in C were analysed by Western blotting . H Representative images of cells treated as in C , D before being fixed and immunostained with antibodies against Lamp2 and p62 . Scale bar :

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Figure 3D Cells treated as in C were analysed by Western blotting.H Representative images of cells treated as in C,D before being fixed and immunostained with antibodies against Lamp2 and p62. Scale bar: 20µm


Figure 4A Fibroblasts stably expressing GFP - TFEB were treated 4OHT to induce senescence ( or EtOH control ) . Cells were lysed and subject to Western blotting for antibodies as indicated . B Quantification of A . ( n = 4 independent experimental repeats ) C Quantification of A . ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . B and C : analysed by 2 - tailed , unpaired T - Test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . D Western blot of GFP immunoprecipitation of control ( EtOH ) and senescent ( 4OHT ) cells expressing GFP or GFP - CDK4 and FLAG - TFEB . H Fibroblasts stably expressing GFP - TFEB were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were lysed and subject to Western blotting . I Quantification of H ( n = 4 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were lysed and subject to Western blotting . K , L Quantification of J ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) M Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were fixed and immunostained with antibodies against p62 and Lamp2 , Scale bar : 20µm . O Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were incubated with serum - free media overnight which was collected and subject to ELISA analysis for IL - 6 ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

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Figure 4A Fibroblasts stably expressing GFP-TFEB were treated 4OHT to induce senescence (or EtOH control). Cells were lysed and subject to Western blotting for antibodies as indicated. B Quantification of A. (n=4 independent experimental repeats) C Quantification of A. (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. B and C: analysed by 2-tailed, unpaired T-Test * p<0.05; ** p<0.01; *** p<0.001.D Western blot of GFP immunoprecipitation of control (EtOH) and senescent (4OHT) cells expressing GFP or GFP-CDK4 and FLAG-TFEB.H Fibroblasts stably expressing GFP-TFEB were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. I Quantification of H (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. K, L Quantification of J (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)M Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were fixed and immunostained with antibodies against p62 and Lamp2, Scale bar: 20µm.O Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were incubated with serum-free media overnight which was collected and subject to ELISA analysis for IL-6 (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)