Fig. 4

BET inhibition and depletion repress the expression of RCAN1.4. a-b MDA-MB-231 cells and BT549 cells were treated with various concentrations of BRD4 inhibitor JQ1 for 24 h or 500 nM JQ1 for the indicated times. The RCAN1.4 mRNA levels were quantified using qRT–PCR(a). The cell lysates were prepared for immunoblots (b). c-d MDA-MB-231 and BT549 cells were transiently transfected with BRD4 siRNA for 48 h. The RCAN1.4 and BRD4 mRNA levels were quantified using qRT–PCR(c). The cell lysates were prepared for immunoblots (d). Blue star, RCAN1.1; Red closed circle, RCAN1.4. e BRD4 binding pattern in the promoter regions and in the SE regions of RCAN1.4. The cells were treated with DMSO or JQ1. The data were retrieved from Gene Expression Omnibus (GSE63581). f-g MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against BRD4. The association with the SE region (f) and promoter region (g) of RCAN1.4 was quantified by qPCR. An isotype-matched IgG was used as a negative control. h-i MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against H3K27ac. The association with the SE region (h) and promoter region (i) of RCAN1.4 was quantified by qPCR. j MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against H3K4me1. The association with the SE region of RCAN1.4 was quantified by qPCR. k-n Luciferase reporter assay of RCAN1.4 promote activity and E3 super-enhancer activity in MDA-MB-231 cells and BT549 cells treated with 200 nM JQ1 for 24 h (k, l), or treated with BRD4 siRNA for 48 h (m, n). Error bars represent mean ± SD, n = 3 biological independent samples. * P < 0.05, **P < 0.01. The P value in a, c, m, n was determined by one-way analysis ANOVA with Dunnett’s multiple comparisons test, the P value in f, g, h, i, j was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in k, l was determined by a two-tailed unpaired Student’s t test. Data were representative of three independent experiments