Figure 6. SERPINA3K inhibits the H₂O₂-induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways.

(A) The rMC-1 cells were treated with 400 µM H₂O₂ in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca²⁺] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H₂O₂-induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H₂O₂ for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H₂O₂. The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H₂O₂ for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P<0.01, under H₂O₂ exposure from 1.5 h to 4 h; the U73122 and SERPINA3K-treated cells versus the BSA-treated cells. * P<0.01, versus control cells. # P<0.01, the cells treated with H₂O₂, m-3M3FBS and SERPINA3K versus the cells treated with H₂O₂ and SERPINA3K. & P<0.01, versus control cells exposed to H₂O₂ for 0 min.