Fig. 3. Cpd16 ameliorates H₂O₂-induced apoptosis and programmed necrosis in osteoblasts.

Primary murine osteoblasts (A–H), human osteoblasts (I–N) or the MC3T3-E1 murine osteoblastic cells (O, P) were pretreated (for 2 h) with Cpd16 (5/25 μM), or plus H₂O₂ (400 μM) stimulation; the caspase-3/-9 activities (A, B, I) were measured; apoptosis-associated proteins were measured (C); cell apoptosis was examined by Annexin V flow cytometry (D, J, results were quantified) and the nuclear TUNEL staining (E, K, and O, results were quantified) assays, with cell viability measured through CCK-8 assays (F, L); CyPD-ANT1-p53 mitochondrial complexation and the expression were shown (G, M), and cell necrosis measured through measuring LDH releasing (H, N, P). *P < 0.05 versus “C” cells. ^#P < 0.05 versus cells with H₂O₂ stimulation but “Veh” pretreatment. Scale bar = 100 μm.