Fig. 6

BMAL1 directly bound to p53 and regulated its protein stability. A, B HBEs were incubated for 48 h after transfection with control siRNA or specific siRNA against BMAL1 and treated with carbon particles (20 μg/ml) or PM2.5 (200 μg/ml) for 24 h, after which p53 mRNA levels were measured by RT-qPCR. (A) n = 6. B n = 5. *P < 0.05 versus the control group (Student’s t-test). C, D After transfection with control or specific siRNA against BMAL1 for 48 h, HBEs were incubated with CHX for 4 h or timing gradients, after which levels of p53 protein were measured by western blot analysis. Changes in relative density of p53 to GAPDH were presented. E After transfection with control or specific siRNA against BMAL1 for 48 h, HBEs were incubated with MG-132 for 2 h, after which the levels of p53 protein were measured. Changes in relative blot density of p53 to GAPDH were presented. F–H HBEs were treated with carbon particles or PM2.5; or transfected with control or specific siRNA against BMAL1 and carbon particles or PM2.5 for 24 h. Immunoprecipitation assay was accomplished with anti-p53 or anti-IgG antibody and the endogenously precipitated complexes were detected with anti-ubiquitin antibody. I, J HEK293T cells were cotransfected with Flag-p53 and control vector; or HA-BMAL1 and control vector; or Flag-p53 and HA-BMAL1 expression vector for 72 h respectively. The co-immunoprecipitation assay was performed with anti-Flag, anti-HA or anti-IgG antibody and the precipitated complexes were detected with anti-HA or anti-Flag antibodies. K, L HBEs were used for the immunoprecipitation experiments. The experiments were carried out with anti-p53, anti-BMAL1 or anti-IgG antibody and the endogenously precipitated complexes were detected with anti-p53 or anti-BMAL1 antibody. Data are mean ± SEM of n individual experiments, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 between indicated conditions (One-way ANOVA followed by the Bonferroni’s test)