Figure 3. circHIPK3 silencing potentiates H2O2-induced death and apoptosis in human osteoblasts. OB-6 human osteoblastic cells were transfected with the lentiviral circHIPK3 shRNA (“sh-circHIPK3-a/b”, with non-overlapping sequences) or control shRNA lentivirus (“sh-C”), following puromycin selection the stable cells were established. 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 “sh-circHIPK3-a” lentivirus or “sh-C” lentivirus for 24h, and then treated with hydrogen peroxide (H2O2, 250 μM) and cultured for the applied time periods, relative circHIPK3 expression, cell viability and death were tested by qPCR (H), MTT (I), and LDH release assay (J), respectively. Expression of the listed proteins was quantified and normalized to the loading control protein (β-) Tubulin (D). Quantified values were mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Veh” treatment of “sh-C” cells. #P < 0.05 vs. H2O2 treatment of “sh-C” cells. Experiments were repeated five times, with similar results obtained. Bar=100 μm (E and G).