Fig. 3

ABT-263 induced autophagy of the macrophages by blocking Bcl-2 binding to Beclin-1. a-c To avoid significant cell death becoming a confusion factor, we took the max dose at which over 80% cells were viable after 24 h as the proper concentration in the further experiments. Cell Counting Kit-8 (CCK-8) was used to quantify viable mouse peritoneal macrophages after treated by different concentrations of ABT-263, 3-MA, Rapamycin for 24 h, respectively. * indicated P < 0.05 compared to the first column of each figure. d-f To explore the effective dose of ABT-263 on inhibiting Bcl-2, the relative mRNA expressions of Bcl-2, BAX, Beclin-1 of the mouse peritoneal macrophages treated with five incremental concentrations of ABT-263, which were measured by real-time RT-PCR. g-h The Co-immunoprecipitation (Co-IP) of the whole cell lysates of the peritoneal macrophages. The cell lysates were firstly subjected to immunoprecipitation using Beclin-1 antibody and detected by Western blot using Bcl-2 antibody. The result showed Bcl-2/Beclin-1 complex was decreased (white arrow) when the cells were treated by ABT-263. The mouse serum was used as the Negative Control (NC). i-l The proteins, Lc3, p62, Trem-2 and Tubulin-α of the mouse peritoneal macrophages were detected by western blotting after treated by 1 μM LPS combined with 2.5 μM ABT-263, 100 μM 3-MA, and 1.0 μM Rapamycin for 24 h respectively. The data in the figures j, k, i, represented the mean ± S.D. Significant differences between groups were indicated as * P < 0.05 compared to the Ctrl group, n = 4