Figure 2. Forced overexpression of circHIPK3 alleviates H2O2-induced death and apoptosis in human osteoblasts. OB-6 human osteoblastic cells were infected with circHIPK3-expressing lentivirus (“LV-circHIPK3”) or control lentivirus (with empty vector, “Vec”), following puromycin selection stable cell lines were established (“OE-circHIPK3-L1/2”). Cells were treated with hydrogen peroxide (H2O2, 250 μM) and cultured for the applied time periods, relative circHIPK3 expression was tested by qPCR assay (A); Cell viability (B), cell death (C), cell apoptosis (D–F) and mitochondrial depolarization (G) were tested by the assays mentioned in the text, and results were quantified. The primary human osteoblasts were infected with “LV-circHIPK3” or “Vec” for 24h, then treated with hydrogen peroxide (H2O2, 250 μM) and cultured for the applied time periods, relative circHIPK3 expression and cell death were tested by qPCR (H) and LDH release (I) assays, respectively; Cell apoptosis was tested by TUNEL staining (J) and Annexin V-FACS (K) assays. Expression of the listed proteins was quantified and normalized to the loading control protein (β-) Tubulin (D). “MW” stands for molecular weight (Same for all Figures). Quantified values were mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Veh” treatment of “Vec” cells. #P < 0.05 vs. H2O2 treatment of “Vec” cells. Experiments were repeated five times, with similar results obtained. Bar=100 μm (E, G and J).