Fig 2: 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.

Fig 3: Executioner caspases activity is induced by Listeria monocytogenes infection in viable host cells that inhibits intracellular growth.A HeLa cells were treated with staurosporine (STS, 0.1 μg/ml) or infected with virulent Lm (WT, strain 10403S), listeriolysin O deficient Lm (ΔLLO) or Salmonella enterica serovar Typhimurium (SL1344) at a MOI of 1 for 1 h before extracellular bacteria were removed by gentamicin treatment. After further culture, the cells were lysed at indicated time points before caspase activity in the lysates was assessed using the chromogenic dye DEVD-pNA. Averages +/− SEM of three independent experiments are shown. HeLa cells infected with indicated bacteria as above +/− DEVD (20 μM), zVAD (20 μM) or STS (0.1 μg/ml) treatment were lysed at the indicated time points and their lysates were assessed by western blot for cleaved caspases-9 and -7 (Cl. casp9 and casp7), as well as the caspase-3-cleavage fragment of Parp1 (cl. parp1, B) or cleaved caspase-3 (cl. casp3) after 16 h (C). α−tubulin served as loading control. HeLa cells were infected with Lm WT as above or left non-infected (non-inf) for 16 h +/− TNF-α (10 ng/ml) before measuring caspase-9, caspase-3 and caspase-7 cleavage (D), as well as activation of caspase 8 (E) by immunoblot. In all panels, representative blots of three independent experiments are shown. DEVDase activity (F), MTS metabolic activity (G) and LDH release (H) were assessed in HeLa cells infected as above for 16 h with Lm WT, Lm ΔLLO or in uninfected cells +/− TNF-α (10 ng/ml) +/− zVAD (20 μM) treatment. Average +/− SEM of three independent experiments is shown. I HeLa cells were infected with Lm WT as above +/− DEVD-fmk (20 μM), zVAD (20 μM) or TNF-α (10 ng/ml) treatment. At indicated times, cells were lysed, and Lm were enumerated by CFU assay. Average +/− SEM of three independent experiments is presented. Asterisks indicate significant differences to untreated controls. P-values are * < 0.05, ** < 0.01 and *** < 0.005.

Fig 4: POLRMT shRNA provokes apoptosis activation in NSCLC cells.The primary human NSCLC cells (pNSCLC1, pNSCLC2, and pNSCLC3) or established A549 cells, expressing the applied POLRMT shRNA (“sh-POLRMT-S1/S2/S3/S4”) or scramble non-sense control shRNA (“scr”), were established and cultured for applied time periods, the relative caspase-3 activity (A), caspase-PARP cleavages (B), ssDNA contents (ELISA OD, C), and mitochondrial depolarization (JC-1 assays, D, F) were tested, and results were quantified and normalized. Cell apoptosis was tested by nuclear TUNEL staining assays (E, G), with results quantified. The primary human lung epithelial cells (“Lung epi”), expressing the sh-POLRMT-S1 (“sh-POLRMT”) or scramble non-sense control shRNA (“scr”), were established. POLRMT mRNA was tested by qRT-PCR assays (H). Cells were further cultured for applied time periods, and cell viability and apoptosis were tested by CCK-8 (I) and TUNEL staining (J) assays, respectively, and results were quantified. “pare” stands for parental control cells. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “scr” cells. n.s. stands for non-statistical difference (I, J).The experiments were repeated five times with similar results obtained. Scale bar = 100 μm (D, E).

Fig 5: NSD3 KO by CRISPR/Cas9 exerts tumor-suppressive functions in pancreatic cancer cells.Single stable PanCa-1 primary pancreatic cancer cells, bearing the lenti-CRISPR/Cas9 construct encoding NSD3 sgRNA (“koNSD3”) or the lenti-CRISPR/Cas9 empty vector (“koC”), were established; expression of NSD3 mRNA and protein was examined by qRT-PCR (A) and western blotting (B) assays, respectively. Cells were further cultured for applied time periods, cell viability (CCK-8 assays, C), proliferation (nuclear EdU staining assays, D), as well as cell migration (“Transwell” assays, E) and invasion (“Matrigel Transwell” assays, F) were tested, with results quantified); caspase-3 activity (G), mitochondrial depolarization (JC-1 staining assays, H) and cell apoptosis (TUNEL staining assays, I) were tested as well. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “koC” cells. Experiments in this figure were repeated five times with similar results obtained. Scale bar = 100 μm (D, H).