Fig 1.
(a) Cadmium induced ROS generation dose-dependently in rPT cells. (b) Intracellular ROS levels in rPT cells after 12-h cadmium (2.5 μM) treatment in the absence or presence of NAC (100 μM). DCF fluorescence was measured using a flow cytometer with FL-1 filter. Confocal microscopy of cadmium-induced MPTP opening. (c) Representative confocal images of cadmium-induced rPT cells. (d) Quantification of calcein fluorescence. Calcein fluorescence values were quantified relative to the control, where the fluorescence value was set at 100%. (e) Representative electron micrographs of rPT cell mitochondria following cadmium exposure (×6600 magnification). Figure shows membrane disruption (thin arrows), swelling, and damaged cristae (thick arrows). Fluorescence results are expressed as mean fluorescence intensity, and are the mean ± SD of three separate experiments, each performed in triplicate (n = 9). *P < 0.05, **P < 0.01 as compared to control.
Fig 2.
Confocal microscopy of MPTP opening after 12-h cadmium (2.5 μM) treatment in the absence or presence of CsA (5 μM).
(a) Representative confocal images of cadmium-induced rPT cells. (b) Quantification of calcein fluorescence. The calcein fluorescence values were quantified relative to the control, where the fluorescence value was set at 100%. (c) Flow cytometry detection of the apoptosis rate after 12-h cadmium (2.5 μM) treatment in the absence or presence of CsA (5 μM) with annexin V–FITC/PI staining. (d) Percentage of apoptotic cells. Results are the mean ± SD of three separate experiments, each performed in triplicate (n = 9). **P < 0.01 as compared to control.
Fig 3.
Cadmium induced mitochondrial cyt c release to the cytoplasm and subsequent caspase-9 and caspase-3 activation in rPT cells.
(a, c) Representative western blots of cyt c, cleaved caspase-9, and cleaved caspase-3. (b, d) Quantitative analysis of cyt c, cleaved caspase-9, and cleaved caspase-3 western blots; grayscale of the control was set at 1. Quantitative analysis was performed with images from three independent experiments (mean ± SD, n = 3). *P < 0.05, **P < 0.01, and ##P < 0.01 as compared to control.
Fig 4.
Cadmium induced BNIP-3 expression and cytoplasmic AIF and Endo G translocation to the nucleus after 12-h cadmium treatment.
(a, c, e) Representative images of BNIP-3, AIF, and Endo G western blots. (b, d, f) Quantitative analysis of BNIP-3, AIF, and Endo G; grayscale of the control was set at 1. (g) Cadmium treatment (12 h) triggered AIF nuclear translocation dose-dependently. rPT cells were stained with anti-AIF antibodies and Alexa Fluor 488–labeled goat anti-rabbit IgG. AIF nuclear translocation was evaluated under fluorescence microscopy with DAPI staining. Scale bar: 50 μm. Effects of BNIP-3 silencing on changes in BNIP-3 expression and cytoplasmic AIF and Endo G translocation to the nucleus after 12-h cadmium treatment in the absence or presence of BNIP-3 small interfering RNA (siRNA). (h, j, l) Representative images of BNIP-3, AIF, and Endo G western blots. (i, k, m) Quantitative analysis of BNIP-3, AIF, and Endo G; grayscale of the control was set at 1. Results are from three independent experiments (mean ± SD, n = 3). **P < 0.01, #P < 0.05, and ##P < 0.01 as compared to control.
Fig 5.
Effects of Z-VAD-FMK on cadmium-induced rPT cell apoptosis.
(a) Representative images of cleaved caspase-3 western blot after 12-h cadmium treatment in the absence or presence of Z-VAD-FMK. (b) Quantitative analysis of cleaved caspase-3; grayscale of the control was set at 1. (c) Flow cytometry assessment of the rPT cell apoptosis rate after 12-h cadmium treatment with/without Z-VAD-FMK. (d) Percentage of apoptotic cells. Results are expressed as the mean ± SD of three separate experiments, each performed in triplicate (n = 9). **P < 0.01 as compared to control.
Fig 6.
Effect of Z-VAD-FMK on BNIP-3 expression and cytoplasmic AIF translocation to the nucleus after 12-h cadmium treatment.
(a) Representative images of BNIP-3 western blot. (b) Quantitative analysis of BNIP-3; grayscale of the control was set at 1. (c) Twelve-hour cadmium treatment with/without Z-VAD-FMK triggered AIF nuclear translocation. rPT cells were stained with anti-AIF antibodies and Alexa Fluor 488–labeled goat anti-rabbit IgG. AIF nuclear translocation was evaluated under fluorescence microscopy with DAPI staining. Scale bar: 50 μm. Results are from three independent experiments (mean ± SD, n = 3). **P < 0.01 as compared to control.
Fig 7.
Effects of BNIP-3 silencing on cadmium-induced apoptosis, mitochondrial cyt c release to the cytoplasm, and changes in caspase-9 and caspase-3 expression.
(a) Flow cytometry assessment of the rPT cell apoptosis rate after 12-h cadmium and/or BNIP-3 siRNA treatment. (b) Percentage of apoptotic cells. (c, e) Representative images of cyt c, caspase-9, and caspase-3 western blots. (d, f) Quantitative analysis of cyt c, caspase-9, and caspase-3; grayscale of the control was set at 1. Results are from three independent experiments (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, and ##P < 0.01 as compared to control.