Figure 1.
Figure 1. Alternative splicing of Bnip3 in cancer cells. (A) Endogenous mRNA levels of Bnip3 full length (Bnip3FL) and Bnip3 spliced variant deleted for exon 3 (Bnip3Δex3) by qPCR in neonatal cardiac myocytes (NCMC), Panc-1, HCT-116, and MCF-7 cells. Data were obtained from at least n = 4–7 independent experiments for each condition tested. Data are expessed as mean ± SD (error bars). *, statistically different from Bnip3FL of primary NCMC; **, statistically different from Bnip3Δex3 of NCMC. (B) Western blot analysis of endogenous PDK2 proteins in Panc-1 cells and NCMC under normoxia and hypoxia conditions. The filter was probed with an antibody directed against PDK2 (46 kD). GAPDH served as a loading control for the Western blot. (C) Endogenous mRNA expression levels of Bnip3FL and Bnip3Δex3 isoforms in NCMC transfected with vector alone control (CTRL) or PDK2 eukaryotic expression plasmid. Data were obtained from at least n = 3 independent experiments for each condition tested. *, statistically different from CTRL. (D) Western blot analysis of endogenous PDK2 protein in Panc-1 cells treated with 1–5 mM DCA. The filter was probed with an antibody directed against PDK2 (46 kD). β-Actin served as a loading control for the Western blot. (E) ELISA for PDK2 kinase activity in Panc-1 cells treated with 5 mM DCA or knocked down for PDK2. Data were obtained from at least n = 4 independent cell culture for each condition tested. *, statistically different from CTRL. (E, inset) Western blot analysis of endogenous PDK2 protein in Panc-1 cells in the absence or presence of siRNA-PDK2. (F) Real-time qPCR analysis of relative Bnip3Δex3 and Bnip3FL mRNA expression levels in Panc-1 cells. Cells were treated with 1–5 mM DCA. Data were obtained from at least n = 3 independent experiments for each condition tested. *, statistically different from CTRL in the absence of DCA. (G) Real-time qPCR analysis of relative Bnip3Δex3 mRNA and Bnip3FL mRNA in Panc-1 cells treated with pyruvate. Panc-1 cells were treated with 5 mM DCA or followed by knockdown of PDK2 with siRNA directed against PDK2 in the absence or presence of 0.1–2 mM pyruvate. Data were obtained from at least n = 3–4 independent cell cultures for each condition tested. *, statistically different from CTRL; NS, not statistically different from each other in the indicated group; **, statistically different from CTRL in the absence of pyruvate. (H) Real-time PCR analysis of Bnip3FL and Bnip3Δex3 isoforms in NCMC, Panc-1, HCT116, and MCF-7 cells subjected to hypoxia (HPX) for 18 h. Data were obtained from at least n = 3–4 independent experiments for each condition tested. *, statistically different from myocytes CTRL for Bnip3FL isoform; †, not statistically different from myocytes CTRL for Bnip3FL isoform; **, statistically different from myocytes CTRL for Bnip3Δex3 isoform. (I) Cell viability of cancer cells during hypoxia; shown are representative epifluorescence images of cells stained with vital dyes calcein-AM and ethidium homodimer-1 to visualize live (green) and dead (red) cells, respectively, as we described previously. Bar, 40 µm. (J) Histogram depicting quantitative data from I. Data were obtained from at least n = 3–4 independent experiments counting ≥500 cells from n = 3 glass coverslips for each condition tested. *, statistically different from myocytes CTRL.

Alternative splicing of Bnip3 in cancer cells. (A) Endogenous mRNA levels of Bnip3 full length (Bnip3FL) and Bnip3 spliced variant deleted for exon 3 (Bnip3Δex3) by qPCR in neonatal cardiac myocytes (NCMC), Panc-1, HCT-116, and MCF-7 cells. Data were obtained from at least n = 4–7 independent experiments for each condition tested. Data are expessed as mean ± SD (error bars). *, statistically different from Bnip3FL of primary NCMC; **, statistically different from Bnip3Δex3 of NCMC. (B) Western blot analysis of endogenous PDK2 proteins in Panc-1 cells and NCMC under normoxia and hypoxia conditions. The filter was probed with an antibody directed against PDK2 (46 kD). GAPDH served as a loading control for the Western blot. (C) Endogenous mRNA expression levels of Bnip3FL and Bnip3Δex3 isoforms in NCMC transfected with vector alone control (CTRL) or PDK2 eukaryotic expression plasmid. Data were obtained from at least n = 3 independent experiments for each condition tested. *, statistically different from CTRL. (D) Western blot analysis of endogenous PDK2 protein in Panc-1 cells treated with 1–5 mM DCA. The filter was probed with an antibody directed against PDK2 (46 kD). β-Actin served as a loading control for the Western blot. (E) ELISA for PDK2 kinase activity in Panc-1 cells treated with 5 mM DCA or knocked down for PDK2. Data were obtained from at least n = 4 independent cell culture for each condition tested. *, statistically different from CTRL. (E, inset) Western blot analysis of endogenous PDK2 protein in Panc-1 cells in the absence or presence of siRNA-PDK2. (F) Real-time qPCR analysis of relative Bnip3Δex3 and Bnip3FL mRNA expression levels in Panc-1 cells. Cells were treated with 1–5 mM DCA. Data were obtained from at least n = 3 independent experiments for each condition tested. *, statistically different from CTRL in the absence of DCA. (G) Real-time qPCR analysis of relative Bnip3Δex3 mRNA and Bnip3FL mRNA in Panc-1 cells treated with pyruvate. Panc-1 cells were treated with 5 mM DCA or followed by knockdown of PDK2 with siRNA directed against PDK2 in the absence or presence of 0.1–2 mM pyruvate. Data were obtained from at least n = 3–4 independent cell cultures for each condition tested. *, statistically different from CTRL; NS, not statistically different from each other in the indicated group; **, statistically different from CTRL in the absence of pyruvate. (H) Real-time PCR analysis of Bnip3FL and Bnip3Δex3 isoforms in NCMC, Panc-1, HCT116, and MCF-7 cells subjected to hypoxia (HPX) for 18 h. Data were obtained from at least n = 3–4 independent experiments for each condition tested. *, statistically different from myocytes CTRL for Bnip3FL isoform; †, not statistically different from myocytes CTRL for Bnip3FL isoform; **, statistically different from myocytes CTRL for Bnip3Δex3 isoform. (I) Cell viability of cancer cells during hypoxia; shown are representative epifluorescence images of cells stained with vital dyes calcein-AM and ethidium homodimer-1 to visualize live (green) and dead (red) cells, respectively, as we described previously. Bar, 40 µm. (J) Histogram depicting quantitative data from I. Data were obtained from at least n = 3–4 independent experiments counting ≥500 cells from n = 3 glass coverslips for each condition tested. *, statistically different from myocytes CTRL.

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