Fig 2: CBP suppression prevented rhHMGB1 from reducing PP2A activity and facilitating MKP-1 degradation. (A) THP-1 cells and primary MPM cells were pretreated with SGC-CBP30 (4 µM) for 2 h, followed by rhHMGB1 (100 ng/ml) treatment for 1 h. Whole cell lysates were immunoblotted using anti-CBP, anti-p-PP2A(Y307), anti-methyl-PP2A-Ca/ß, anti-PP2A-Ca/ß, anti–p-MKP-1(S359) or anti–MKP-1 antibodies, respectively. (B) THP-1 cells were transfected with CBP shRNA or negative control shRNA, and 72 h after transfection, cells were stimulated with or without rhHMGB1 (100 ng/ml) for 1 h. Cell lysates were subjected to immunoblotting with indicated antibodies. THP-1 cells were treated the same as in (A), and cell lysates were immunoprecipitated with anti-PP2A-Ca/ß then immunoblotted with anti–PME-1 and anti-PTPA antibodies (C). The mRNA expression levels of MKP-1 were detected by RT-PCR (D). THP-1 cells were pretreated with SGC-CBP30 (4 µM) for 2 h, followed by rhHMGB1 (100 ng/ml) for 1 h in the presence or absence of MG132 (20 µM). Cell lysates were immunoprecipitated using anti–MKP-1 antibody, followed by immunoblotting using antibody against ubiquitin (Ub) (E). Cells were transfected with CBP shRNA, and 72 h after transfection, then treated with or without rhHMGB1 (100 ng/ml) for 1 h in the presence or absence of MG132 (5, 10, or 20 µM). Western blot analysis was performed using anti-p-MKP-1 (S359) or anti-MKP-1 antibodies (F). Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., no significance.

Fig 3: CBP bromodomain inhibitor SGC-CBP30 prevented rhHMGB1-induced inflammatory response by rescuing PP2A/MKP-1. THP-1 cells were pretreated with SGC-CBP30 (4 µM), and then with okadaic acid (100 mM) (A) or RO 31-8220 (5 µM) (B) for 1 h, followed by treatment with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Cells were transfected with Flag-PP2A (C) or Flag-MKP-1 (D) and pretreated with SGC-CBP30 (4 µM) for 2 h, followed by treatment with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Cells were transfected with PP2A shRNA (E) or MKP-1 shRNA (F), followed by pretreatment with SGC-CBP30 (4 µM) for 2 h and then stimulation with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.