Fig. 6.
Effects of U-0126 and pifithrin (PFT)-α on doxorubicin (Dox)-induced apoptosis pathway in H9c2 cells. Cells were pretreated with or without 20 μM U-0126 or PFT-α for 1 h and then cultured in the presence or absence of 1 μM doxorubicin for 24 h. A: cell apoptosis was measured by annexin V-fluorescein isothiocyanate (FITC) and PI using FACSCalibur flow cytometry. Histograms show the percentages of early (annexin V-FITC positive and PI negative) and late (annexin V-FITC positive and PI positive) apoptotic cells in control and doxorubicin-treated cells with and without U-0126 and PFT-α treatment. B: mitochondrial damage was measured by changes in mitochondrial membrane potentials (ΔΨm) using fluorescent JC-1 under fluorescence microscopy. Histograms show the ratios of normal mitochondrial membrane potential (ΔΨm) and collapsed ΔΨm in control and doxorubicin-treated cells with and without U-0126 and PFT-α pretreatment. C: whole cell lysates were used to measure p-ERK1/2 (p-ERK), p-p53, Bax, Bcl-2, PUMA-α, caspase-9, cleaved caspase-3, and PARP by Western blot analysis. β-actin was used as an equal loading control. D: nuclear translocation of p-ERK1/2 and p-p53 was studied by indirect immunofluorescence using fluorescein-conjugated secondary antibodies (p-ERK1/2, green; and p-p53, red). Cell nuclei were counterstained with DAPI. Representative images are shown from 3 experiments. E and F: nuclear proteins were processed for Western blot analysis to detect p-ERK1/2 and p-p53. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control; †P < 0.05, compared with doxorubicin.