Extended Data Fig. 2: Inhibition or deletion of BRD4 inhibits mESC cardiac differentiation.
From: A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation
(a) Expression of Myh6, Nkx2-5, and Tnnt2 at day 7 of mESCs cardiac differentiation treated with JQ1 (100 nM) starting day 5 of differentiation (n = 3 biologically independent samples). (b) TNNT2+ cells quantified by flow cytometry at d9 of mESC differentiation in CMV-CreERT2;Brd4flox/flox cells treated with 4-hydroxytamoxifen (TAM), JQ1 (250 nM) or MZ3 (500 nM) starting at day 5 (n = 3 biologically independent samples; FACS gating strategy in Supplementary Fig. 1). (c-d) Immunofluorescence of TNNT2 at day 9 of mESC to CM differentiation in wild type cells treated with JQ1 (100 nM) or vehicle starting at day 5. (e-f) Immunofluorescence of BRD4 in vehicle (ethanol, e-e′′) and TAM (f-f′′) treated undifferentiated mESCs. (g) Volcano plot showing RNA-seq from CMV-CreERT2;Brd4flox/flox (TAM vs. vehicle [VEH]) mESCs and gene ontology analysis of downregulated and upregulated genes (see Methods for details). (h) Heatmap showing expression of select transcription factor and muscle structural protein genes from RNA-seq in CMV-CreERT2;Brd4flox/flox (TAM vs. VEH) mESC-derived cardiac tissues (day 10; TAM or VEH added at day 5). In all graphs, error bars represent ±1 SEM. For a, all comparisons are made relative to 0 nM compound for each gene; * represents p < 0.0493, ** represents p < 0.0085 (two-tailed unpaired t test). For b, all comparisons are made relative to VEH for each condition; * represents p < 0.0188 (two-tailed unpaired t test). Scale Bars = 100 µm (c, d, e, e′, e′′, f, f′, f′′).
