Research Paper Volume 12, Issue 10 pp 9240—9259

Figure 1. LPS promotes senescence of macrophages with increased expression of BRD4 via NFκB pathway activation. Cultures of THP-1 monocyte-derived macrophages were prepared and lipopolysaccharide (LPS, 1 μg·ml⁻¹) was used to induce senescence. (A) Senescent THP-1 monocyte-derived macrophages induced by LPS were detected by senescence-associated β-galactosidase staining (SA-β-Gal staining). Scale bar, 50 μm. The quantification of the SA-β-gal positive cells is presented in the scatter plot. (B) Representative western blot and statistical data showing the protein levels of senescence markers p53, p21, and p16, with or without LPS. Actin was used as the loading control. (C) Real-time polymerase chain reaction (RT-PCR) was used to assess the expression of senescence-associated secretory phenotype (SASP) genes. (D) BRD2, BRD3, BRD4, pNF-kB and NF-kB levels were evaluated by western blotting. Actin was used for normalization. (E) mRNA levels of BRD2, BRD3, and BRD4 in THP-1 macrophages with or without LPS. (F) Immunofluorescence analysis of THP-1 macrophages with or without LPS stained for BRD2 (red), BRD3 (red), BRD4 (green), p16 (green), and Nuclei (DAPI, blue) were analyzed by confocal microscopy. (G) THP-1 macrophages were transfected with NF-κB-specific siRNA, followed by 1μg/ml LPS stimulation for 24 hours. Western blotting analysis and quantification of pNF-κB, NF-κB and BRD4 protein expression in THP-1 macrophages. Actin was used for normalization. The data all represent measurement data presented as the mean ± SD. The two groups were statistically analyzed using independent sample t-test. The experiment was repeated three times. Significant differences among the different groups are indicated as *p <0.05 vs. control; **p <0.01 vs. control; ***p <0.001 vs. control.