Figure 3

(+)-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).