A . Simple western analysis ( Peggy Sue ) and quantifications ( right panels ) of mouse lung epithelial cells shows increased ph - EZH2 ( T311 ) , ph - TAK1 , myosin activity ( ph - MLC2 ) and POL2 - K7m levels in AAV - mediated TGFβ1 over - expression . Note , increased ph - EZH2 and POL2 - K7m levels are attenuated by the EZH2 inhibitor GSK126 , whereas ph - TAK1 and ph - MLC2 levels cannot be rescued by GSK126 . Quantifications ( right panels ) show violin plots , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = non - significant , Kruskal - Wallis / Dunn ' s . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD B-ORGANISM O

A. Simple western analysis (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph-EZH2 (T311), ph-TAK1, myosin activity (ph-MLC2) and POL2-K7m levels in AAV-mediated TGFβ1 over-expression. Note, increased ph-EZH2 and POL2-K7m levels are attenuated by the EZH2 inhibitor GSK126, whereas ph-TAK1 and ph-MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant, Kruskal-Wallis/Dunn's. Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


B . Representative of 3D computed tomography ( CT ) reconstruction of the lung from control , AAV - TGFβ1 and GSK126 - treated AAV - TGFβ1 mice ( green : lung tissue , red : airways , and region - of - interest ( ROI ) : blue ) . Insets show µCT slices in the middle of the lung from respective mice . Note , GSK126 attenuates TGFβ1 - induced lung injury . Quantification ( right panel ) shows mean intensity of ROIs from the whole lung ( violin plots , * p = 0 . 0385 , * * p = 0 . 0012 , ANOVA / Holm - Sidak ' s ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

O O O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O B-ORGANISM O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD B-ORGANISM O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O B-SMALL_MOLECULE O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD B-ORGANISM O

B. Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV-TGFβ1 and GSK126-treated AAV-TGFβ1 mice (green: lung tissue, red: airways, and region-of-interest (ROI): blue). Insets show µCT slices in the middle of the lung from respective mice. Note, GSK126 attenuates TGFβ1-induced lung injury. Quantification (right panel) shows mean intensity of ROIs from the whole lung (violin plots, *p = 0.0385, **p = 0.0012, ANOVA/Holm-Sidak's). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


D . Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph - EZH2 ( scale bars 100 µm ) , pro - SFTPC as a marker for alveolar type 2 epithelial cells ( scale bars 50 µm ) and quantifications ( right panels ) show percentage of KRT5 + pods area per 10X field and percentage of pro - SFTPC + cells per 20X field ( * p < 0 . 05 , unpaired t - test ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

O O B-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD B-ORGANISM O

D. Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph-EZH2 (scale bars 100 µm), pro-SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5+ pods area per 10X field and percentage of pro-SFTPC+ cells per 20X field (*p < 0.05, unpaired t-test). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


( a ) MEFs stably expressing EGFP - Parkin were exposed to CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis ( IB ) with antibodies to the indicated proteins . The asterisk indicates a nonspecific band .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band.


( b ) SH - SY5Y cells were exposed to CCCP ( 10 μM ) for the indicated times and then subjected to immunoblot analysis .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O

(b) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis.


( c ) MEFs stably expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope . The fluorescence of EGFP - Parkin was monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


( a , c ) MEFs ( a ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively .

O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.


( a , c ) HeLa cells ( c ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively .

O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.


( e ) NIH 3T3 cells stably expressing EGFP - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , after which whole - cell homogenates as well as cytosolic , ER and mitochondrial fractions were prepared and subjected to immunoblot analysis . Scale bar ( a - d ) , 10 μm .

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(e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.


( a ) MEFs stably expressing EGFP - tagged wild - type ( WT ) or T240R mutant forms of Parkin as well as FLAG - tagged VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP was also monitored directly .

O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O

(a) MEFs stably expressing EGFP-tagged wild-type (WT) or T240R mutant forms of Parkin as well as FLAG-tagged VAP-A were incubated in the absence or presence of CCCP (30 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP was also monitored directly.


( b ) MEFs as in a were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis .

O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O

(b) MEFs as in a were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis.


( c ) MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP - Parkin was also monitored directly .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(c) MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP-Parkin was also monitored directly.


( d ) MEFs stably expressing EGFP - Parkin were incubated for 12 h in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) as indicated and then subjected to immunoblot analysis . Scale bar ( a , c ) , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

(d) MEFs stably expressing EGFP-Parkin were incubated for 12 h in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) as indicated and then subjected to immunoblot analysis. Scale bar (a,c), 10 μm.


( b ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were treated with CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . Scale bar , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm.


( c ) MEFs stably expressing mCherry - tagged Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O

(c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


( a ) MEFs stably expressing FLAG - Parkin and either EGFP - Bcl - 2 or EGFP - Bcl - xL were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O

(a) MEFs stably expressing FLAG-Parkin and either EGFP-Bcl-2 or EGFP-Bcl-xL were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


( c ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of Bcl - 2 , Bcl - xL , Bcl - 2 / xL or Bcl - xL / 2 were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of Bcl-2, Bcl-xL, Bcl-2/xL or Bcl-xL/2 were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


( a ) MEFs stably expressing EGFP - FKBP38 ( N402K ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - FKBP38 ( N402K ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.


( b ) MEFs stably expressing EGFP - FKBP38 ( N402K ) , mCherry - FKBP38 and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG ( M2 ) . The fluorescence of EGFP - FKBP38 ( N402K ) and mCherry - FKBP38 was also monitored directly .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(b) MEFs stably expressing EGFP-FKBP38(N402K), mCherry-FKBP38 and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG (M2). The fluorescence of EGFP-FKBP38(N402K) and mCherry-FKBP38 was also monitored directly.


( c ) MEFs stably expressing FLAG - Parkin and either EGFP - FKBP38 or EGFP - FKBP38 ( N402K ) were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( d ) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in ( c ) . Data are expressed relative to the corresponding control value ( CCCP 0 h ) and are means ± s . d . , from three independent experiments . * * P0 . 01 ( Student ' s t test ) .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).


( e ) MEFs stably expressing EGFP - Bcl - 2 ( H235R ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - Bcl - 2 ( H235R ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each set .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) MEFs stably expressing EGFP-Bcl-2(H235R) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-Bcl-2(H235R) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.


( g ) HeLa cells stably expressing FLAG - Parkin were transiently transfected with vectors for KikGR - FKBP38ΔN47 or KikGR - FKBP38ΔN47 ( N402K ) , exposed to UV , incubated in the absence or presence of CCCP ( 10 μM ) for 4 h , and then monitored for red and green KikGR fluorescence . Scale bar ( a , b , e , g ) , 10 μm .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(g) HeLa cells stably expressing FLAG-Parkin were transiently transfected with vectors for KikGR-FKBP38ΔN47 or KikGR-FKBP38ΔN47(N402K), exposed to UV, incubated in the absence or presence of CCCP (10 μM) for 4 h, and then monitored for red and green KikGR fluorescence. Scale bar (a,b,e,g), 10 μm.


( a ) Wild - type ( Mfn2 + / + ) and Mfn2 knockout ( Mfn2 - / - ) MEFs stably expressing mCherry - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c . The fluorescence of mCherry - Parkin was also monitored directly .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly.


( b ) MEFs as in a but expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . The asterisk indicates a nonspecific band .

O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.


( c ) Wild - type MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) or nocodazole ( 10 μM ) for 6 h as indicated , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c , to FLAG and to FKBP38 . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) Higher - magnification views of the boxed areas in ( c , e ) . Wild - type MEFs stably expressing FLAG - Parkin and EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 10 h and then subjected to immuno - electron microscopic analysis with antibodies to GFP . Black arrowheads indicate the mitochondria . White arrowheads indicate small vesicle .

O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O 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-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O

(c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle.


( f ) Wild - type ( Fkbp38 + / + ) and Fkbp38 knockout ( Fkbp38 − / − ) MEFs stably expressing FLAG - Parkin and either EGFP or EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then stained with allophycocyanin ( APC ) - labelled annexin V and propidium iodide ( PI ) for flow cytometric analysis of the percentage of apoptotic cells ( annexin V + and PI + cells ) . Data are means ± s . d . , from three independent experiments .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments.


( 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(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 .

O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O

(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 .

O O O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-GENEPROD B-ORGANISM O O O O O

(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 .

O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-GENEPROD O O

(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 .

O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(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 ) .

O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O

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 ) .

O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O

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 ) .

O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O

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 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 .

O O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( 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 .

O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 ) .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM O O O O

(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 .

O O O O O O O O O O O B-DISEASE B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE B-DISEASE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-DISEASE O O O O O O O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O B-DISEASE O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O

(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 .

O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE B-DISEASE O O O B-GENEPROD B-ORGANISM O

(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 ) .

B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE B-DISEASE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O

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 ) .

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE B-DISEASE O O O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

(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 ) .

O O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE B-DISEASE O O O O O O O O O O O O O O O O

(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 .

O O O O O O O B-TISSUE I-TISSUE O O O O B-DISEASE O

(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 .

O O O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE B-DISEASE O O O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

(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 O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( B ) SCs were FACS sorted 4 months after the last TM injection ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells .

O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O

(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 .

O O O O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O

(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 .

O O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O B-TISSUE O O O O O O B-ORGANISM O

(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 .

O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O

(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 .

O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O

(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 .

O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-GENEPROD B-ORGANISM O O O O O O O

(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 ) .

O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O O

(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 ) .

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(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 ) .

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(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 .

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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 .

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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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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 ) .

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 ) .

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(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 ) .

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(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 .

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(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.


( 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O

(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 .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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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 Suz12 (Q) binding on selected mitochondrial genes.


ChIP - qPCR was performed to show the enrichment of H3K27 me3 ( R ) binding on selected mitochondrial genes .

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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 .

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(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.


( 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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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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 .

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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. by


An equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production ( G ) were measured and normalized with cell numbers . N = 3 mice .

An 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O

(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 .

O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O

(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 .

O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O

(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 .

O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O

(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 .

O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(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 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(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 O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O

(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 O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

O O O O O O O B-GENEPROD B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O 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-ORGANISM O

(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 .

O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O

(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 .

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(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 .

O O O B-CELL_TYPE O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O

(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 .

O O O B-CELL_TYPE O O O O O B-GENEPROD B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O

(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 .

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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 .

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 O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR - GFP and H2B - RFP were treated as described above and images were acquired once per hour for 12 h ( A ) . For one representative experiment among three , the mean ± SEM of the average area of high CALR dots ( normalized to the control at each timepoint ) of quadruplicates is shown ( B ) . Values are depicted as the area under the curve ± SD of

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM B-DISEASE B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR-GFP and H2B-RFP were treated as described above and images were acquired once per hour for 12 h (A). For one representative experiment among three, the mean ± SEM of the average area of high CALR dots (normalized to the control at each timepoint) of quadruplicates is shown (B). Values are depicted as the area under the curve ± SD of triplicates


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1 - GFP and H2B - RFP images were acquired every hour for 24 h ( D ) . For one representative experiment among three , the mean ± SEM of the green fluorescence intensity in the nucleus ( normalized to the control at each timepoint ) of quadruplicates is depicted ( E ) . For each cell , the speed of nuclear release ( difference of HMGB1 nuclear green fluorescence intensity between two time points ) was calculated . Values are depicted as the average speed of the nuclear release ± SD of

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O 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-SUBCELLULAR O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1-GFP and H2B-RFP images were acquired every hour for 24 h (D). For one representative experiment among three, the mean ± SEM of the green fluorescence intensity in the nucleus (normalized to the control at each timepoint) of quadruplicates is depicted (E). For each cell, the speed of nuclear release (difference of HMGB1 nuclear green fluorescence intensity between two time points) was calculated. Values are depicted as the average speed of the nuclear release ± SD of quadruplicates


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated for 6 , 12 or 24 h and ATP was stained with quinacrine ( G ) . The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions . For one representative experiment among three the mean ± SD of quadruplicate assessments is

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated for 6, 12 or 24 h and ATP was stained with quinacrine (G). The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions. For one representative experiment among three the mean ± SD of quadruplicate assessments is shown


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated with MTX or DACT as described above for 6 h . Then medium was refreshed and 24 h later , type I interferon response was assessed by transferring the supernatant on HT29 MX1 - GFP reporter cells lines cells for additional 48 h . Human type 1α interferon ( IFNα1 ) was also added on the cells as an additional positive control . Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions ( I ) . The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_LINE B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated with MTX or DACT as described above for 6 h. Then medium was refreshed and 24 h later, type I interferon response was assessed by transferring the supernatant on HT29 MX1-GFP reporter cells lines cells for additional 48 h. Human type 1α interferon (IFNα1) was also added on the cells as an additional positive control. Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions (I). The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depicted


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated as mentioned above for 6 h and then medium was refreshed . Twenty - four hours later , cells were collected and surface - exposed calreticulin ( CALR ) was stained with an antibody specific for CALR . DAPI was used as an exclusion dye and cells were acquired by flow cytometry ( K ) . The percentage of CALR + cells among viable ( DAPI - ) ones are depicted . The mean ± SEM of six independent experiments is

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O 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-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated as mentioned above for 6 h and then medium was refreshed. Twenty-four hours later, cells were collected and surface-exposed calreticulin (CALR) was stained with an antibody specific for CALR. DAPI was used as an exclusion dye and cells were acquired by flow cytometry (K). The percentage of CALR+ cells among viable (DAPI-) ones are depicted. The mean ± SEM of six independent experiments is depicted


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit , then normalized to control . The mean ± SEM of four independent experiments is shown .

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit, then normalized to control. The mean ± SEM of four independent experiments is shown.


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h . Concentration of secreted ATP in the supernatant was quantified with a luciferase - based bioluminescence kit . The mean ± SD of quadruplicates from one representative among three experiments is depicted .

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h. Concentration of secreted ATP in the supernatant was quantified with a luciferase-based bioluminescence kit. The mean ± SD of quadruplicates from one representative among three experiments is depicted.


Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin ( DACT ) ( 0 . 25 , 0 . 5 or 1 µM ) for 6 h . Thapsigargin ( THAPS ) at 3 µM was used as a positive control . After fixation , cells were stained with phospho - eIF2α ( Ser51 ) specific antibody followed by an AlexaFluor - 647 secondary antibody , nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy . Images were segmented , analyzed and the red cytoplasmic fluorescence intensity was measured . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

B-ORGANISM B-DISEASE B-CELL_LINE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O

Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS cells stably expressing ATF4 - reporter were treated as described above for 12 h . The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS stably expressing ATF6 - GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS cells stably expressing venus in frame with alternatively spliced XBP1 ( sXBP1 ) were treated as described above for 12 h . De novo expressed venus was measured intracellular . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS wild - type and knock out for eIF2α kinases 1 , 2 , 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3 - mediated eIF2α phosphorylation or with 1 µM DACT for 6 h . After fixation , cells were stained for peIF2α as described above and cytoplasmic intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS CALR - RFP were injected subcutaneously ( s . c ) in the flank of nu / nu mice . Tumors were further injected with PBS ( Ctr ) ( n = 5 ) , 0 . 5 mg / kg tunicamycin ( TM ) for 6 h ( n = 3 ) or 0 . 5 mg / kg DACT for 6 h ( n = 4 ) or 24 h ( n = 3 ) . Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor - 647 secondary antibody and counterstained with Hoechst 33342 . Two slices per tumor were imaged for their DAPI , RFP and Cy5 signals . Out - of - focused images were removed from the dataset leading to the analysis of the following conditions : 8 x Ctr , 5 x TM , 8 x DACT 6 h and 6 x for DACT 24 h . PeIF2α was quantified measuring Cy5 intensity in the cytoplasm ( K , L ) and CALR translocation by measuring the coefficient variation ( CV ) of the RFP signal in the cytoplasm ( M , N ) . Representative images of peIF2α are shown for Ctr , TM and DACT at 6 h ( K ) , whereas images of CALR are shown for Ctr and DACT at 24

B-CELL_LINE B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O

U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h