Figure 3.

HBO1 shRNA provokes OS cell apoptosis. Stable primary OS cells (pOS-1 and pOS-2), as well as the established OS cell lines (U2OS and MG63) that expressed HBO1 shRNA (shHBO1-a/shHBO1-b) or the scramble control shRNA (“shC”) were established and cultured for applied time periods, caspase activation (A, B and J), single strand DNA (ssDNA) contents (C), and mitochondrial depolarization (by recording JC-1 green monomers, D and I) were tested; Cell apoptosis was tested by nuclear TUNEL staining (E and K) and Annexin V FACS (F) assays. Stable pOS-1 cells with shHBO1-a or shC were treated with z-DEVD-fmk (50 μM), z-VAD-fmk (50 µM) or vehicle control (0.1% of DMSO), and cultured for 96h. Cell viability and death were tested by CCK-8 (G) and Trypan blue staining (H) assays, respectively. For nuclear TUNEL staining assays, five random views of a total 1, 000 cells per each condition were included to calculate the average TUNEL ratio (% vs. DAPI, same for all TUNEL assays). “Pare” stands for the parental control OS cells. The data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “sh-C” cells. ^#P < 0.05 vs.“DMSO” treatment (G and H). The experiments were repeated five times with similar results obtained. Scale bar = 100 µm (D, E, I and K).