Fig. 5: MZT2B silencing impedes viability, proliferation, migration, and induces cell cycle arrest in primary and immortalized 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”). Relative mRNA levels of MZT2B and MZT2A were determined (A). MZT2B and MZT2A protein expression was assessed by immunoblot analysis (B). Following a cultivation period, the nuclear EdU incorporation, indicative of DNA synthesis and cell proliferation, was quantified (C). Cellular viability was evaluated via CCK-8 assay optical density measurements (D). Colony formation was measured using clonogenic assays (E). Cell cycle distribution was analyzed by flow cytometry (F). Cell migration (G) and invasion (H) were quantified using Transwell chambers. The analogous experiments were conducted in additional primary human NSCLC cells (pNSCLC2 and pNSCLC3), as well as the immortalized A549 cell line, engineered to maintain stable expression of lentivirus-delivered kdMZT2B-sh2 or shC. The relative mRNA levels of MZT2B (I) and MZT2A (J) were determined. The nuclear EdU incorporation (K) and CCK-8 optical density measurements (L) were quantified. Cell migration (M) was assessed via Transwell chambers. “Ctrl” denotes parental control cells. Error bars represent the mean ± standard deviation (SD), with statistical significance (*P < 0.05 when comparing to “shC” cells). “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.