Fig. 5: ORC6 silencing/KO leads to apoptosis activation in glioma cells.

P1 primary glioma cells with the described lentiviral ORC6 shRNA (“shORC6-s1 and shORC6-s2”), the Cas9 construct plus the lentiviral CRISPR/Cas9-ORC6-KO construct (“koORC6”), or the lentiviral scramble control shRNA plus CRISPR/Cas9 control construct (“shC+Cas9-C”), were established and underwent further cultivation for specified durations; Cytosolic lysates were obtained for the assessment of caspase-3/caspase-9 activity (A, B), analysis of apoptosis-related protein expression using Western blotting assays (C), and quantification of Cytochrome C levels utilizing an ELISA kit (D). Cell apoptosis was evaluated using nuclear TUNEL staining (E, F) and Annexin V FACS (G) assays, while cell death was examined through trypan blue staining assays (H). P1 glioma cells with control lentiviral shRNA (“shRNA”) or ORC6-targeting shRNA (“shORC6-s1”) were treated with or without the respective caspase inhibitors (each at 50 μM) for specific time periods; Cell viability and death were assessed using CCK-8 (I) and Trypan blue staining (J) assays, respectively. Other primary glioma cells, P2/P3 or A172 established cells with the lentiviral scramble control shRNA (“shC”) or the lentiviral ORC6 shRNA (“shORC6-s1”) were established and cultured for indicated time periods, the caspase-3 activity (K) and apoptosis (nuclear TUNEL staining, L) were tested. The primary human astrocytes (“Astrocytes1/2”) were modified to express either “shORC6-Sq1” or “shC,” and expression of ORC6 mRNA tested (M); An equal number of the astrocytes were cultured for designated time periods, cell viability, proliferation and apoptosis were tested by CCK-8 (N), nuclear EdU staining (O) and TUNEL staining (P) assays respectively, with results quantified. Data were presented as mean ± standard deviation (SD, n = 5). “Pare” denoted the parental control cells. *P < 0.05 compared to “shC+ Cas9-C”/“shC” cells. “n. s.” indicates non-statistical difference (P > 0.05). The experiments were conducted five times (biological repeats), yielding consistent results. Scale bar = 100 μm.