EMBO/SourceData huggingface dataset:
Figure 4(B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue.(C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated.(D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69).(E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student's t-test.
Figure 1B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25 µm).E. ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from MCs and IPF-MCs in the co-culture system. Note a stronger αSMA response in IPF-MCs (mean + s.d., n = 3 biological replicates from 6 MCs and 6 IPF-MCs donors, **p = 0.0014, ****p < 0.0001, Nonlinear Regression followed by ANOVA/Sidak's).G, H. 72 h after substitutions in the co-culture, αSMA and E-cad levels of MCs and AECs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E-cad levels (mean + s.d., ns = non-significant, ***p < 0.001, **p < 0.01, n = 3 biological replicates with 6 MCs donors, repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett´s posthoc test).
Figure 2B. Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co-culture medium upon apical TGFβ1 stimulation (p-adj < 0.05, t-test), with examples of profibrotic secreted proteins (red dots indicating significantly upregulated proteins, blue dots indicating significantly downregulated proteins, and grey dots indicating no significant change in protein expression levels).H. Representative H3K27me3 and EZH2 immunofluorescence images and box plots (minimum, first quartile, median, third quartile and maximum) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1-injured AECs in co-culture with MCs (n = 5 biological replicates with >50 cells per experiment, scale bars 50 µm, *p < 0.05, unpaired t-test).
Figure 3G. EZH2-deleted AECs (sgEZH2) reintroducing empty vector (EV), T311 wildtype (WT), a phosphorylated-deficient T311A, or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes. Vehicle and TGFβ1 treated AECs (sgNEG + vehicle / TGFβ1) were used as control. mRNA levels are normalised to HPRT1 expression. (mean + s.d., n = 4 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis/Dunn's).
Figure 4B. Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co-culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph-EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non-significant, ***p < 0.001, Kruskal-Wallis/Dunn's).E. ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.
Figure 5C, D. ChIP-qPCR shows increased occupancy of (C) POL2-S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2-S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Figure S5B (mean + s.d., n = 3 biological replicates). E, F. ChIP-qPCR shows increased occupancy of (E) POL2-S5p at promoters and (F) POL2-S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Figure S5C (mean + s.d., n = 3 biological replicates).
Figure 6B. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1-induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown.E. qPCR analysis of profibrotic genes in MCs co-culture with AECs shows that depletion of IPO9 in TGFβ1-injured AECs blocks the fibrotic crosstalk with MCs. Data show mRNA levels of profibrotic genes normalised to S26 (mean + s.d., n = 3 biological replicates with 5 MCs donors, *p < 0.05, ***p < 0.001, ANOVA/Tukey's).
Figure 7A. 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.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.
f1(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.(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.
f2(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.(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.
f3(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.(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.
f4(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.
f5(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.
f7(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.(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.(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.
f8(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.(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.
Figure 1(B-C) Immunofluorescent (IF) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors. Scale bar = 100 µm.(D) Representative images of Ctrl, Heterozygous (Het) and YY1cKO newborn (P0) mice or embryos at E18.5 day.(E) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test. The lung from YY1cKO pup sank whereas Ctrl floated.(F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates.(H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm.The above harvested Dps were subjected to IF staining for MyHC together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).The above harvested Dps were subjected to IF staining for Troponin T (TnT) together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).(K) Representative images of H&E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18.5 day. Scale bar = 400 µm (left) or 50 µm (right).The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of MyHC+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb muscles were subjected to IF staining for TnT together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of TnT+ fibers per area are shown on the right (n=3 mice, each).The above harvested limb (N) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 100 µm (N) Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.The above harvested diaphragm (O) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 50 µm (O). Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01 ***P<0.001.
Figure 2(B) SCs were FACS sorted 3 days after the last TM injection and cultured for 1.5 days; RT-qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells.(C) IF staining for Pax7 and YY1 on freshly isolated FISCs or (D) single myofibers from EDL muscles or (E) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells. Scale bar =100 µm in (C) or 50 µm in (D-E).(F) Representative FACS plots. About 100,000 cells from 2-month-old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post-TM injection. The percentage of SCs was shown. (n=3 mice, each).(G) H&E staining was performed on the injured TA muscles collected from the designated times post-CTX injection to visualize the degree of regeneration. Scale bar = 100 µm.(H) IF staining for eMyHC on the TA muscles 5 days post-CTX injury. Quantifications of the number of eMyHC+ fibers are shown on the right (n=3, each). Scale bar = 50 µm.(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.(J) Cross-section area (CSA) of individual myofibers in TA muscles 4 weeks after injury was measured. The distribution for CSA was calculated (n=3 mice).(K) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice.Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of Pax7+ SCs per area are shown on the right (n=3 mice, each).Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of MyoD+ SCs per area are shown on the right (n=3 mice, each).(N) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). Scale bar = 100 μm.(P) H&E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury. Scale bar = 400 μm (left) or 100 μm (right).(Q) Representative images of above TA muscles 7 days after second injury.(R) Quantifications of the weight above TA muscles from three pairs of littermate mice.(S) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice. Quantifications of the number of eMyHC+ fibers per area are shown on the right (n=3 mice, each). Scale bar = 100 μm.(T) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7+ SCs per area are shown on the right (n=3 mice, each). Scale bar = 100 μm. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.
Figure 3(B) SCs were FACS sorted 4 months after the last TM injection; RT-qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells.(C) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration.(D) Representative images of limb muscles (Tibialis anterior, TA; Gastrocnemius, Gas; Quadruple, Quad) from Ctrl or YY1dKO mice. (E) Quantifications of muscle weight from three pairs of littermate mice.(F) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection.(G) H&E or (H) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Quantifications of the numbers of MyHC+ fibers per area of TA or Dp muscles are shown on the right (n=3 mice, each). Scale bar = 100 µm.(I) Collagen1 or (J) Masson's Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Scale bar = 100 µm.(K) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6-month-old Ctrl and YY1dKO mice was calculated (n=3 mice, each).(L) Representative FACS plots showing the percentage of SCs sorted from 6-month-old Ctrl and YY1dKO mice (n=3 mice, each).(M) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each).EDL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.