Fig. 3: SLC5A3 depletion provokes apoptotic death in NSCLC cells.

Patient-derived pCan-1 primary NSCLC cells, bearing the designated SLC5A3 lentiviral shRNA (shSLC5A3-S1 and shSLC5A3-S2) or the lenti-CRISPR/Cas9-SLC5A3-KO construct (koSLC5A3), were established; The control cells were with the scramble non-sense lentiviral shRNA plus the lenti-CRISPR/Cas9-KO empty vector (“shC+koC”). After culturing for the designated hours, cell death (Trypan blue ratio, A), the relative caspase-3 activity (B), expression of apoptosis-related proteins (Western blotting assays, C), ssDNA contents (ELISA OD, D) and mitochondrial depolarization (JC-1 green monomers accumulation, E) were tested. Cell apoptosis was examined by measuring the ratio of TUNEL-positively stained nuclei (F) and the number of Annexin V-positive cells (FACS assays, G). Patient-derived primary NSCLC cells (pCan-2/3, derived from two other patients), the immortalized NSCLC lines (A549 and H460), the primary lung epithelial cells (“pEpi”) or the immortalized BEAS-2B cells, stably expressing the SLC5A3 shRNA (shSLC5A3-S1) or the scramble non-sense lentiviral shRNA (shC) were established. After culturing for the designated hours, cell death (by measuring Trypan blue ratio, H and K), the relative caspase-3 activity (I), and apoptosis (by measuring the percentage of the TUNEL-positive nuclei, J and K) were tested similarly. “Pare” indicated the parental control cells. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 versus “Pare”/“shC” group. “N. S.” indicated no statistical difference (P > 0.05). Each single experiment was repeated for five times. Scale bar = 100 μm.