Figure 2. The PI3K/AKT signaling pathway is involved in GPR30-induced HMGB1 secretion in CAFs. (A) HMGB1 mRNA expression and (B) protein levels using paired fibroblasts isolated from immortalized cells and (C) HMGB1 secretion in the supernatant of nine immortalized NFs and CAFs. Treatment with G1 (1 μM) and TAM (10 nM) for 30 min, with or without pretreatment with G15 (1 μM) in CAFs, and then detection of HMGB1 mRNA (D) and secretion (E). All values are shown following normalization against the internal control β-actin (* P < 0.05, ** P < 0.01). Bars represent the mean range of HMGB1 less than 0.05 compared with the matched NF-CM treatment. Detection of the expression of HMGB1 by western blot (F) and qRT-PCR (G, H) after infection with lentivirus carrying GPR30-shRNA. Cells were cultured with CM supplemented with G1 (1 μM) or TAM (10 nM) in the presence or absence of G15 (1 μM) and LY294002 (10 μM) for 24 h, and HMGB1 was detected by ELISA (I) and western blotting (J). Total Akt and phosphorylated Akt were measured by western blot (J, K).