Fig 2: BRD4 inhibitors efficacy. (A) Cell viability assay; the bar graph shows percentage cell viability count in treatments with JQ1 after 12 h treatments. (B) bar graph shows percentage cell viability count in treatments with RVX208 after 12 h treatments. Inhibition of BRD4 function and target genes expression (C,D) Immunofluorescence images for the effect of RVX208 and JQ1 on the cellular localization of NF-κB in response to indicated treatments. HUVECs are stained with anti-NF-κB antibody (red) and DAPI (blue). Two-way ANOVA with Bonferroni’s post-test used. Values are mean ± SD of three biological.

Fig 3: Expression analysis of BRD4 isoforms in HUVECs. (A) Bar graph showing relative mRNA levels of BRD4-Total normalized to house-keeping gene GAPDH. BRD4-L and BRD4-S isoforms normalized first to the house-keeping gene and then to BRD4-Total. (B) Western blot image and the corresponding relative quantification bar graph showing the protein expression of BRD4-L and BRD4-S. Immunoblot is stripped and reprobed with β-actin antibody. The β-actin (~42kDa) is used as a loading control. The band intensities of BRD4-L and BRD4-S are normalized to β-actin. (A,B) Two-way ANOVA with Bonferroni’s post-test used; values are mean ± SD of two biological replicates (** = p < 0.01 and * = p < 0.05).

Fig 4: BRD4 inhibitor efficacy. (A) Bar graph showing relative mRNA levels of BCL2 and HMOX-1 after normalization to house-keeping gene GAPDH, in DMSO control, TNF-α-only, RVX208, and RVX208, followed by TNF-α treatment (left panel). (B) DMSO control, TNF-α-only, JQ1, and JQ followed by TNF-α treatment (right panel). One-way ANOVA with Tukey’s post-test used. Values are mean ± SD of three biological replicates (** = p < 0.01, * = p < 0.05 and ns = non significant).

Fig 5: Midkine inhibition in HUVECs with BRD4 inhibitors. (A) Bar graph showing relative mRNA levels of midkine in control and TNF-α treatments (6, 12, 24, 48 and 72 h), RVX208 treatment and pretreatment with RVX208 followed by TNF-α (6, 12, 24, 36, 48, and 72 h). (B) Bar graph showing relative mRNA levels of midkine in control and TNF-α treatments (6, 12, 24, 48 and 72 h), JQ1 treatment and pretreatment with JQ1 followed by TNF-α (6, 12, 24, 36, 48, and 72 h). One-way ANOVA with Tukey’s post-test used; values are mean ± SD of three biological replicates (*** = p < 0.001) Data are shown after normalization to housekeeping gene GAPDH. (C,D) Western blot image showing the protein expression of midkine in cell supernatant (upper panel) in control, TNF-α treatments (36, 48, and 72 h), and pretreatment with RVX208 (C) or JQ1 (D) followed by 48 and 72 h of TNF-α treatment. Ponceau staining (lower panel) of the Western blot membrane shows a band of unknown identity at ~55 kDa as a loading control.