Myogenic differentiation is inhibited by BETi compounds. C2C12 myoblasts were cultured in GM for 2 days and then switched to DM for 3 days. Cultures were treated with BET inhibitors; 10 µM (+)-JQ1, 1 µM PFI-1, and 1 µM Bromosporine at the same time as switching to DM. Untreated (DM only), DMSO-treated, and 10 µM (−)-JQ1-treated cultures were used as negative controls. (a) Myogenic differentiation was assessed by immunofluorescence (IF) staining for Myosin Heavy Chain (MHC), and (b) quantified using Myogenic and Fusion Indices. (c) MHC and MYOG protein expression was determined by Western blot. IMR-90 fibroblasts were treated with doxycycline (DOX) to induce MYOD1 expression and then switched to DM to induce differentiation. Cultures were treated with; 100 nM (+)-JQ1, 1 µM PFI-1, and 1 µM Bromosporine, or controls at the same time as switching to DM. (D) Myogenic differentiation was assessed by (d) MHC IF staining and (e) Myogenic and Fusion Indices. (f) MHC and MYOD1 protein expression was determined by Western blot. (g) Primary mouse satellite cells and (h) primary human skeletal muscle cells were treated with 1 µM (+)-JQ1 or (−)-JQ1 negative control. Myogenic differentiation was assessed by MHC IF. All microscopy images were taken at 10 × magnification, scale bars indicate 50 µm. Blots were cropped for conciseness and clarity. All values are mean + SD, n = 4 representative fields of view, **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test, and comparisons to the (−)-JQ1 control group reported.

Inhibition of myogenic differentiation by (+)-JQ1 is dose-dependent. C2C12 cultures were treated with (+)-JQ1, or (−)-JQ1 negative control enantiomer, over a range of concentrations (1 nM to 10 µM) and myogenic differentiation assessed by (a) MHC IF staining, and (b) quantified using Myogenic and Fusion Indices. (c) MHC and MYOG protein levels were determined by Western blot. (d) Myh1, Myog and Ckm mRNA levels were determined by RT-qPCR. All images were taken at 10× magnification, scale bars indicate 50 µm. Blots were cropped for conciseness and clarity. All values are mean +/− SD, n = 4 representative fields of view for microscopy, n = 3 for RT-qPCR, **P < 0.01, ***P < 0.001 (unpaired t-test comparing (+)-JQ1 and (−)-JQ1 at each time point).

(+)-JQ1 inhibits the cell cycle by G1 arrest in differentiating myoblasts. C2C12 cultures were treated with (+)-JQ1, or the (−)-JQ1 inactive enantiomer, over a range of concentrations (1 nM to 10 µM) and cell viability assessed by (a) MTS assay, and (b) nuclei counting. Cell cycle progression was assessed by EdU incorporation assay. (c) C2C12 cultures were switched to DM and treated with 1 µM (+)-JQ1, (−)-JQ1, DMSO or untreated (DM only). Cells were fixed at 12, 24, 48 and 72 hours after treatment and pulsed with EdU 2 hours before fixation. EdU positive nuclei were stained with Alexa Fluor 555 and cultures immunostained for MHC. (d) The percentage of EdU positive nuclei was determined by cell counting. (e) Changes in the number of nuclei over time, and in response to compound treatment were determined by counting nuclei. (f) The effect of 1 µM (+)-JQ1 on cell cycle progression was determined by flow cytometry analysis of DNA content, (g) and the proportions of nuclei in G1, S and G2 phases quantified. Images were taken at 10 × magnification, scale bars indicate 50 µm. Values are mean +/− SD, n = 4 representative fields of view for microscopy, n = 6 for MTS data, n = 3 cultures for flow cytometry (30,000 singlet events counted per sample), *P < 0.05, **P < 0.01, ***P < 0.001 (Comparisons between two groups were tested using an unpaired t-test. Comparisons between multiple groups were tested using one-way ANOVA with Bonferroni post hoc test, and the result for the (−)-JQ1 versus (+)-JQ1 comparison reported).

Effect of BET inhibition in growth media. (a) C2C12 myoblasts were cultured in GM and treated after 24 hours with 1 µM (+)-JQ1 or controls; (−)-JQ1 inactive stereoisomer, DMSO, or untreated (GM only). Cells were harvested 24 and 48 hours after treatment. Prior to fixing cells were pulsed with EdU for 2 hours to label proliferating cells. (b) EdU incorporation was visualized by fluorescence microscopy, and (c) the percentage of EdU positive cells determined by cell counting. (d) In parallel, cell viability was assessed by MTS assay. (e) To test the effect of compound treatments prior to the induction of differentiation, C2C12 cells were treated with compounds (as above) in GM for 24 hours and then switched to DM for 3 days. Myogenic differentiation was assessed by MHC IF staining for Myosin Heavy Chain (MHC), and (f) Myogenic and Fusion Indices quantified, and nuclei counted. Similar experiments were further performed to control for cell density. (g,h) Firstly, C2C12 cells were treated for 24 hours in GM, collected by trypsinization and cells counted. Equal numbers of cells (2 × 10⁵ cells per well of a 24 well plate) from each treatment group were re-seeded in DM and cultured for a further three days before fixing. (i,j) Secondly, C2C12 cells were treated in GM for 24 hours, trypsinized, and counted as described above. Equal numbers of cells were re-seeded in GM for an additional 24 hours, before switching to DM for a further 3 days. Images were taken at 10× magnification, scale bars indicate 50 µm. Values are mean +/− SD, n = 4 representative fields of view for microscopy, n = 9 for MTS data, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test, and the result for the (−)-JQ1 versus (+)-JQ1 comparison reported.

BETi treatment inhibits differentiation independently of cell cycle arrest. (a) C2C12 cells were treated with 1 µM (+)-JQ1 for a 24 hour interval starting at day 2, 3 or 4 (corresponding to zero, one or two days in Differentiation Media; DM0, DM1 and DM2). Fresh DM was added at each time point in order to wash out the compound treatments. (b) Myogenic differentiation was assessed by MHC IF staining and compared with DMSO and (−)-JQ1-treated negative controls. (c) Myogenic transcript levels for Myog, Myh1 and Ckm were determined for each treatment protocol by RT-qPCR. Cells treated with GM only were included as an additional undifferentiated control. (d) C2C12 cells were cultured for 3 days in GM in order to reach full confluence and promote maximal cell cycle withdrawal prior to switching to DM for 3 days. Cultures were treated with 1 µM (+)-JQ1 concurrent with the switch to DM. Cultures treated with (−)-JQ1, DMSO, or left untreated (DM only) served as negative controls. Cells were fixed, and myogenic differentiation was assessed by MHC IF staining. (b) Light microscopy images indicate that cells were fully confluent by day 2 in Growth Media (GM2). (c) Changes in the cell cycle markers Ccnd1a (Cyclin D1) and Cdkn1a (p21) were assessed by RT-qPCR, and (d) CCND1A by western blot. All microscopy images were taken at 10× magnification, scale bars indicate 50 µm. Blots were cropped for conciseness and clarity. All values are mean + SD, n = 3, **P < 0.01, ***P < 0.001. The mean of the DMSO group (treated at DM0) was scaled to a value of one. Comparisons between multiple groups were tested using one-way ANOVA with Bonferroni post hoc test, and comparisons to the (−)-JQ1 or GM1 group reported.

BETi treatment induces prolonged inhibition of myogenic differentiation in differentiation conditions. (a) C2C12 cells were treated with 1 µM (+)-JQ1 or 10 µM SB 203580 for a 3 day period in DM and (b) cultures either fixed for MHC IF staining or cultured for an additional 3 days in fresh DM to washout the compounds before fixing. Myogenic differentiation was assessed relative to untreated (DM only), DMSO, and (−)-JQ1 negative controls. (c) Myogenic transcript levels for Myog, Myh1 and Ckm were determined for each treatment protocol by RT-qPCR. Cells treated with GM only were included as an additional undifferentiated control. Images were taken at 10× magnification, scale bars indicate 50 µm. All values are mean + SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. The mean of the DMSO group at day 5 was scaled to a value of one. Comparisons between multiple groups were tested using one-way ANOVA with Bonferroni post hoc test, and comparisons to the DMSO group reported.

Differential expression and function of BET bromodomain proteins during myogenic differentiation. C2C12 cells were cultured in GM for 2 days followed by DM for a further 6 days and cells harvested at the indicated time points. Expression of myogenic factors and BET bromodomain genes was assessed by (a) Western blot, and (b) RT-qPCR. Three antibodies were used against BRD4. Total BRD4 levels were determined using two antibodies; anti-BRD4, which is commercially available, and anti-BRD4 (N) which is an in-house antibody directed against the N-terminus. Phosphorylated BRD4 was measured using anti-BRD4 pS484/488. (c) C2C12 cultures were treated with 100 nM siBrd3, siBrd4 or the negative control siRNA (siCtrl) in GM. After 24 hours, cultures were switched to DM and cultured for a further 48 hours. Myogenic differentiation was assessed by MHC IF staining and (d) quantified using Myogenic and Fusion Indices. Images were taken at 10× magnification, scale bars indicate 50 µm. Blots were cropped for conciseness and clarity. All values are mean + SD, n = 4 representative fields of view for microscopy, n = 3 for RT-qPCR, **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test, and comparisons to the GM1 or siCtrl groups reported.

BRD4 is enriched at the Myog promoter during myogenic differentiation. Chromatin was harvested from undifferentiated C2C12 cells (two days in GM), differentiated cells (2 days in GM, followed by 3 days in DM), and differentiated cells treated with either (−)-JQ1 or (+)-JQ1 (1 μM). Sonicated chromatin was precipitated with anti-H3K27Ac, anti-BRD3, anti-BRD4 or IgG (negative background control) antibodies. Genomic DNA was amplified by qPCR using primers for the Myog promoter or the Sox2 TSS (a putative negative control locus). Enrichment is expressed as the fraction of non-precipitated input samples after subtraction of the background IgG signal. For BRD3, one way ANOVA analysis was significant (P = 0.0399), but no pairwise Bonferroni post test reached significance at the P < 0.05 level. All values are mean + SEM, n = 3 independent experiments, statistical comparisons are to the GM only group, *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA, Bonferroni post hoc test).