Fig. 5: Bromodomain inhibitors suppress the expression of CXCLs by inhibition of transcription factor binding at CXCL super-enhancer and promoter sites.

a, b LSECs were pretreated with iBET151 (0–50 μM) (n = 4, biologically independent samples from two independent experiments) (a) or UMN627 (0–50 μM) (n = 5, biologically independent samples from three independent experiments) (b). CXCL1, 6, and 8 expression levels were assessed by qPCR. Expression levels were normalized to basal conditions and log₁₀ fold change values were plotted. One-way matched-pairs ANOVA analysis was performed with post hoc Dunnett’s multiple comparison correction. Data represented as mean ± SD. c, d ChIP-qPCR assays for BRD4 binding (c, n = 4, biologically independent samples from two independent experiments) or NF-κB binding (d, n = 6, biologically independent samples from three independent experiments) with and without TNFα stimulation was assessed at CXCL super-enhancer and CXCL1 promoter sites after pretreatment with iBET151. One-way ANOVA analysis was performed with post hoc Tukey’s multiple comparison correction. Data represented as mean ± SD. e, f Same experiments were repeated with Celastrol (e, n = 4 biologically independent samples from two independent experiments; f, n = 6 biologically independent samples from three independent experiments). Enrichment for either CXCL1 promoter or CXCL super-enhancer sequence was examined. Sequence enrichment was normalized to input. One-way ANOVA analysis was performed with post hoc Tukey’s multiple comparison correction. Data represented as mean ± SD. There were significant linear trends of decreasing CXCL1, 6, 8 expressions with increasing iBET151 and UMN627 concentrations without and with TNFα, and with increasing iBET151 concentrations without TNFα (p < 0.0001 for all groups). All data were repeated at least three times with similar results.