Fig. 6: MZT2B silencing elicits robust apoptotic cell death in NSCLC cells.

Primary human non-small cell lung cancer (pNSCLC1) cells were engineered to stably express lentivirus-delivered shRNAs targeting MZT2B (“kdMZT2B-sh1” or “kdMZT2B-sh2”, nonoverlapping sequences) or a scrambled non-sense control shRNA (“shC”). Following a cultivation period, Caspase-3 activity (A) and Caspase-9 activity (B) were assessed. The expression of apoptosis-associated proteins (cleaved Caspase-3, cleaved Caspase-9, and cleaved PARP1) was detected b (C). Cytosolic cytochrome c release was measured (D). Cellular apoptosis was examined and quantified via the nuclear TUNEL staining (E) and Annexin V/Propidium Iodide (PI) FACS (F) assays. Overall, cell death was determined by Trypan blue exclusion assay, with results quantified (G). The effect of a Caspase-3 specific inhibitor (z-DEVD-fmk, Cas3i, 400 μM) or a pan-caspase inhibitor (z-VAD-fmk, Casi, 400 μM) on cell death in kdMZT2B-sh2-expressing pNSCLC1 cells or shC cells was investigated (H). Similar experiments were conducted in primary NSCLC cells (pNSCLC2 and pNSCLC3) and the established A549 cell line, following MZT2B knockdown by kdMZT2B-sh2. The nuclear TUNEL staining (I), and overall cell death (J) were similarly assessed. Primary human lung epithelial cells (pEpi1 and pEpi2) were engineered to stably express lentivirus-delivered kdMZT2B-sh2 or shC. Relative mRNA levels of MZT2B (K) and MZT2A (L) were determined. Cell viability was assessed by CCK-8 assay (M). Nuclear TUNEL staining of cell apoptosis assay was carried out, with results quantified (N). “Ctrl” denotes parental control cells. Error bars represent the mean ± standard deviation (SD), with statistical significance (*P < 0.05 when comparing to “shC” cells). ^#P < 0.05 (H). “n.s.” stands for P > 0.05. All experiments presented in this figure were independently replicated five times (n = 5, biological repeats) and consistently yielded similar results. Scale bar = 100 μm.