Fig. 3: Apoptosis activation after YME1L depletion in primary human NPC cells.

The stable pNPC-1 primary NPC cells with the lentiviral YME1L shRNA (“kdYME1L”), the lentiviral CRISPR-YME1L-KO construct (koYME1L), or the lentiviral scramble control shRNA plus the lentiviral CRISPR-KO control treatment (“ctrl”) were established, the exact same number of the above pNPC-1 cells were cultivated for designated hours, Caspase-3 and Caspase-7 activities (A and B), expression of apoptosis-associated proteins (C) and Histone DNA contents (D) were measured. Cell apoptosis was tested via nuclear TUNEL incorporation fluorescence staining assay (E) and Annexin V-PI FACS (F). Cell death was measured by trypan blue staining assays, with results quantified (G). The ctrl pNPC-1 cells or the koYME1L pNPC-1 cells were treated with the antioxidant NAC (0.5 mM) or ATP (1 mM) for designated hours, cell apoptosis (TUNEL assays, H) and death (trypan blue assays, I) were measured. Primary NPC cells that were derived from other patients, pNPC-2/-3/-4, or the primary human nasal epithelial cells (pHNEpC-1 and pHNEpC-2, derived from two donors) were stably transduced with the lentiviral YME1L shRNA (“kdYME1L”) or the lentiviral scramble control shRNA (“kdC”); The exact same number of cells were further cultivated for designated hours, the Caspase-3 activity (J) and apoptosis (TUNEL assays, K and M) were measured similarly. The relative YME1L mRNA expression in the epithelial cells was shown (L). The numerical values were mean ± standard deviation (SD, n = 5). “pare” indicates the parental control cells. *P < 0.05 vs. “pare”/ “kdC” cells. ^# P < 0.05 vs. “koYME1L” cells (H and I). “N. S.” stands for non-statistical difference (P > 0.05). Experiments in this figure were repeated five times, and each time similar results obtained. Scale bar = 100 μm.