Fig 2: Overexpression of miR-1269a variant increases the apoptosis of gastric cancer cells. (A) A representative FCM analysis chart. The effect of miR1269a and miR-1269a variant on apoptosis was examined by FCM analysis. HGC-27 and MGC-803 cells were transfected with miR-control, miR1269a mimics and miR-1269a variant mimics and their inhibitors. Subsequently, the cells were analyzed for apoptotic rate following staining with Muse™ Annexin V Dead Cell reagent. (B) Quantitative data from FCM analysis. Data represent the mean ± standard deviation from three independent experiments. *P<0.05; **P<0.01. (C) Western blot analysis of apoptosis proteins (Bik, Bim and Bak) in HGC-27 and MGC-803 cells, which were transfected with miR1269a mimics and miR-1269a variant mimics. GAPDH served as the loading control. FCM, flow cytometry. Bik, Bcl-2-interacting killer; miRNA, microRNA; Bak, Bcl-2 homologous antagonist/killer.

Fig 3: Evaluation of apoptosis regulating proteins after lexatumumab (1 µg/ml) and DTIC (50 µg/ml) treatment in FEMX-1 and HHMS.The cells were harvested 24 h after treatment exposure and analyzed for various apoptotic markers. (A) Immuno-blot detection of Bcl-w, Bcl-XL, Mcl-1, Bak, Bax, Bik, Bad, BimEL, BimL and BimS expression in FEMX-1 cells. (B) Immuno-blot detection of cIAP-1, cIAP-2, XIAP, survivin and livin in FEMX-1 and HHMS cells. β-actin is used as loading control. Cell lysates were prepared from three independent biological experiments, and representative blots are included in the figure.

Fig 4: ABT-263 reduces endotoxin-induced mucous cell hyperplasia in vivo in a Bik-dependent manner. (A) Experimental outline for testing therapeutic efficacy of ABT-263 in LPS-induced MCH in mice. (B) Representative micrographs of lung tissue sections stained with Alcian-Blue (AB) and H&E from LPS-challenged mice treated with vehicle or ABT-263 (2 mg/Kg) are shown. Quantification of mucous cell numbers per mm BL. (C) Representative micrographs showing activated (cleaved) caspase 3 or Ac-Casp3 (green) among Scgb1a1-positive (red) secretory cells in mouse axial airways. The relative fold-change in the number of ac-Casp3+ secretory cells in LPS-challenged mice treated with vehicle or ABT-263. (D) Representative micrographs showing TUNEL-positivity (green) in Scgb1a1+ (red) secretory cells in mouse axial airways treated with ABT-263 and DAPI-stained nuclei (blue). The relative fold-change in the number of TUNEL+ secretory cells in mice challenged with LPS and treated with vehicle or ABT-263. (E) STAT-1 phosphorylation in HAECs following 0, 15, and 60 minutes of IL-13 treatment. Cropped Western blots are displayed. (F) Bik and Bcl-2 mRNA levels in IL-13 treated STAT1 −/− and STAT1 +/+ MEFs compared with the respective non-treated cells. (G) Immunoprecipitation with anti-Bcl-2 antibodies of proteins extracted from HAECs treated with nothing or ABT-263. Bik levels are reduced in the pull-down while increased in the flow-through of HAECs treated with ABT-263 compared with non-treated controls. Cropped Western blots are displayed. (H) Representative micrographs of lung tissue sections from LPS-challenged mice stained with AB-H&E and quantification of mucous cell numbers per mm BL in mice treated with vehicle or ABT-263 (2 mg/Kg) following LPS challenge. Scale = 20 µM; Data shown as mean ± SEM (n = 5–10 mice/group); *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: CRNDE was involved in epigenetic repression mediated by EZH2, SUZ12, and SUV39H1 on multiple tumor suppressor genes.qPCR was performed to measure CRNDE levels in immunoprecipitates in Bel-100 (a) and Huh-7 cells (b). CRNDE expression levels were displayed as fold changes relative to IgG immunoprecipitate. c RNA pull-down assay was applied to capture the proteins that bound CRNDE in Bel-100, and protein expression levels of HuR, EZH2, SUZ12, and SUV39H1 were determined by WB. HuR and AR were used as positive control. d, e qPCR analysis was performed to measure the relative expression levels of multiple tumor suppressor genes in Bel-100 (d) and Huh-7 cells (e). f, g HCC cells were transfected with lentiviruses encoding siRNAs against EZH2 (f), SUZ12 (g), SUV39H1 (h), or negative control and the protein expression levels of the silenced proteins together with BIK, LATS2, p27KIP1, and CELF2 were determined by WB. i–l HCC cells were transfected with shCRNDE or shNC, and ChIP-qPCR was used to identify the enrichment of SUZ12, SUV39H1, EZH2, H3K27me3, and H3K9me3 at promoter region of BIK (i), LATS2 (j), p27KIP1 (k), CELF2 (l), and GAPDH (m), respectively. The data were shown as Mean ± SD based on at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.