Research Paper Volume 12, Issue 10 pp 9240—9259

Figure 2. BRD4 is involved in macrophage senescence caused by inflammation. THP-1 macrophages were incubated with four different siRNAs for knockdown of BRD4. (A) BRD4 expression was evaluated by western blotting, as shown in the scatter plot. (B) Western blot analysis for BRD2, BRD3, and BRD4 protein expression. Actin was used for normalization. (C) SA-β-gal activity was analyzed after the knockdown of BRD4. The quantification of SA-β-gal positive cells is presented in the scatter plot. (D) Analysis of SASP genes mRNA levels in THP-1 macrophages. The results are presented in the cluster heatmaps. IL-6 and CXCL1 mRNA levels are shown in the histogram on the right. (E) The senescence markers p53, p21, and p16 were analyzed by western blotting. The results are presented in the scatter plot. Actin was used as the loading control. (F) Immunofluorescence images showing BRD4 (green) and p16 (green). The nuclei were counterstained with DAPI (blue). (G) Representative Oil Red O (ORO) staining and statistical data were used to assess lipid uptake. The data all represent measurement data presented as the mean ± SD. The two groups were statistically analyzed using independent sample t-test. One-way ANOVA was used in comparisons among multiple groups, followed by Tukey’s post-hoc test. Significant differences among the different groups are indicated as *p <0.05 vs. LPS; ***p <0.001 vs. LPS; ****p <0.0001 vs. LPS. The experiment was repeated three times.