Fig. 5: JQ1 reduces the transcriptional upregulation of NADPH oxidase subunits. | Nature Communications

Fig. 5: JQ1 reduces the transcriptional upregulation of NADPH oxidase subunits.

From: BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy

Fig. 5

a qRT-PCR analysis of Nox2 (WT, n = 4; mdx, n = 6; mdx+JQ1, n = 8), Nox4 (WT, n = 4; mdx, n = 7; mdx+JQ1, n = 8), p47-phox (WT, n = 3; mdx, n = 6; mdx+JQ1, n = 8) and p67-phox (WT, n = 4; mdx, n = 5; mdx+JQ1, n = 8) mRNAs in TAs from control, vehicle-, and JQ1-treated mice. Data are normalized against HPRT and expressed as the mean ± SD. b Representative images of immunoblot analysis for Nox2 and p67-phox in TAs from control, vehicle-, JQ1-treated mice. GAPDH serves as a loading control. WT animals: n = 3 for each experimental group. c qRT-PCR analysis of NADPH oxidase subunit mRNAs in myofibers isolated from mdx EDL muscles and treated with 200 nM JQ1 for 16 h. Data are normalized against HPRT and expressed as the mean ± SD, n = 3 animals. d qRT-PCR analysis of NADPH oxidase subunit mRNAs in immortalized DMD myoblast cells treated with 200 nMJQ1 for 24 h. Data are normalized against GAPDH and expressed as the mean ± SD, n = 3 immortalized cell lines. e RPKM expression levels of NADPH subunit transcripts in published RNA-Seq dataset for DMD (n = 6) and healthy donors (n = 6). Data are expressed as a mean ± SD. f qRT-PCR analysis of Nox2, Nox4, p47 phox (n = 4 for each experimental group) and p67-phox (n = 3 for each experimental group) mRNAs in C2C12 myotubes cells were pretreated with 200 nM JQ1 and then co-treated with 250 μM H2O2 for 8 h. Data are normalized against GAPDH and expressed as the mean ± SD. g Representative images of immunoblot analysis for Nox2, p67-phox and BRD4 in C2C12 myoblasts treated as in (e). GAPDH serves as a loading control. h qRT-PCR analysis of Nox2 (Ctrl, n = 5; siBrd2, n = 4; siBrd3, n = 3; siBrd4, n = 3), Nox4 (Ctrl, n = 5; siBrd2, n = 3; siBrd3, n = 5; siBrd4, n = 5), p47-phox (Ctrl, n = 5; siBrd2, n = 6; siBrd3, n = 4; siBrd4, n = 4) and p67-phox (Ctrl, n = 5; siBrd2, n = 5; siBrd3, n = 4; siBrd4, n = 3) subunit mRNAs in C2C12 cells in which BRD2, BRD3, BRD4 levels were decreased by siRNAs transfection. Data are normalized against GAPDH and expressed as the mean ± SD. i ChIP assay with BRD2 and BRD4 antibodies in control, vehicle- and JQ1-treated muscles showing recruitment at regulatory regions of Nox2, Nox4, p47-phox and p67-phox genes. Data represent mean ± SD, n = 3 animals A schematic representation below the diagrams shows the region amplified in ChIP. In all panels, statistical significance was determined by using one-way ANOVA followed by Tukey’s post hoc test. a indicates statistical significance compared to the group presented in the first column; b indicates statistical significance compared to the group presented in the second column. *P < 0.05; **P < 0.01; ***P < 0.001.

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