Figure 2.
Figure 2. Knockdown of Bnip3Δex3 isoform sensitizes cancer cells to hypoxia. (A) Schematic depicting the targeting strategy of siRNA for knockdown of endogenous Bnip3Δex3 isoform. siRNA was designed to specifically target sequences spanning the exon 2–exon 4 junction, which is unique to the Bnip3Δex3 isoform. Specificity of the siRNA was described previously. (B) Real-time qPCR analysis of relative mRNA levels of endogenous Bnip3FL isoform. Panc-1 cells were subjected to hypoxia for 18 h in the absence or presence of siRNA-Bnip3Δex3 (50 nM). RNA extracted from the cells was analyzed by real-time PCR. Data were obtained from at least n = 3–4 independent experiments for each condition tested. Data are expressed as mean ± SD (error bars). *, statistically different from control (CTRL); †, not statistically different from HPX. (C) Western blot analysis of Bnip3 proteins in panc-1 cells in the absence or presence of shRNA-Bnip3FL or siRNA-Bnip3Δex3 under the hypoxia condition; the filter was probed with a murine antibody directed against Bnip3 (∼30 kD). β-Actin (∼40 kD) served as a loading control for the Western blot. (D) Real-time qPCR analysis of relative mRNA levels of endogenous Bnip3Δex3 isoform. Panc-1 cells were subjected to hypoxia for 18 h in the absence or presence of siRNA-Bnip3Δex3 (50 nM). RNA extracted from the cells was analyzed by real-time PCR. Data were obtained from at least n = 3–4 independent experiments for each condition tested. *, statistically different from control (CTRL); **, statistically different from hypoxia (HPX). (E) Western blot analysis of Flag-Bnip3Δex3 expression levels in Panc-1 cells. Panc-1 cells were transfected with Flag-Bnip3Δex3 eukaryotic expression vector in the absence or presence of siRNA-Bnip3Δex3. Bnip3Δex3 was detected with the antibody directed against Flag-tag. (F) ROS in Panc1 cells after Bnip3Δex3 knockdown during hypoxia; ROS production was monitored by dihydroethidium (DHE, red fluorescence) staining. Bars, 40 µm. (G) Cell viability of Panc-1, HCT116, and MCF-7 cells after Bnip3Δex3 knockdown during hypoxia. Cell viability was shown by representative epifluorescence images of cells stained with vital dyes calcein-AM and ethidium homodimer-1 to visualize live (green) and dead (red) cells, respectively. Bars, 40 µm. (H) Histogram for quantitative data shown in E. 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 CTRL; †, not statistically different from CTRL.

Knockdown of Bnip3Δex3 isoform sensitizes cancer cells to hypoxia. (A) Schematic depicting the targeting strategy of siRNA for knockdown of endogenous Bnip3Δex3 isoform. siRNA was designed to specifically target sequences spanning the exon 2–exon 4 junction, which is unique to the Bnip3Δex3 isoform. Specificity of the siRNA was described previously. (B) Real-time qPCR analysis of relative mRNA levels of endogenous Bnip3FL isoform. Panc-1 cells were subjected to hypoxia for 18 h in the absence or presence of siRNA-Bnip3Δex3 (50 nM). RNA extracted from the cells was analyzed by real-time PCR. Data were obtained from at least n = 3–4 independent experiments for each condition tested. Data are expressed as mean ± SD (error bars). *, statistically different from control (CTRL); †, not statistically different from HPX. (C) Western blot analysis of Bnip3 proteins in panc-1 cells in the absence or presence of shRNA-Bnip3FL or siRNA-Bnip3Δex3 under the hypoxia condition; the filter was probed with a murine antibody directed against Bnip3 (∼30 kD). β-Actin (∼40 kD) served as a loading control for the Western blot. (D) Real-time qPCR analysis of relative mRNA levels of endogenous Bnip3Δex3 isoform. Panc-1 cells were subjected to hypoxia for 18 h in the absence or presence of siRNA-Bnip3Δex3 (50 nM). RNA extracted from the cells was analyzed by real-time PCR. Data were obtained from at least n = 3–4 independent experiments for each condition tested. *, statistically different from control (CTRL); **, statistically different from hypoxia (HPX). (E) Western blot analysis of Flag-Bnip3Δex3 expression levels in Panc-1 cells. Panc-1 cells were transfected with Flag-Bnip3Δex3 eukaryotic expression vector in the absence or presence of siRNA-Bnip3Δex3. Bnip3Δex3 was detected with the antibody directed against Flag-tag. (F) ROS in Panc1 cells after Bnip3Δex3 knockdown during hypoxia; ROS production was monitored by dihydroethidium (DHE, red fluorescence) staining. Bars, 40 µm. (G) Cell viability of Panc-1, HCT116, and MCF-7 cells after Bnip3Δex3 knockdown during hypoxia. Cell viability was shown by representative epifluorescence images of cells stained with vital dyes calcein-AM and ethidium homodimer-1 to visualize live (green) and dead (red) cells, respectively. Bars, 40 µm. (H) Histogram for quantitative data shown in E. 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 CTRL; †, not statistically different from CTRL.

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