Bim Antibody - #AF0121

Figure 5 DHW-221-triggered mitochondrial apoptosis is linked to Akt-mediated FOXO3a nuclear translocation in A549/Taxol cells. (A, B) Comparison and the expression levels of PI3K/Akt/FOXO3a signaling pathway related proteins in A549 and A549/Taxol cells were determined by Western blot, in the presence and absence of DHW-221 and GDC-0980. Statistical comparisons were performed with unpaired Student’s t-test (n = 3) in Panel (A). *p < 0.05, **p < 0.01 versus A549. Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons in Panel (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control. (C, E) The FOXO3a and p-FOXO3a expressions in the nuclear and cytoplasmic fractions of A549/Taxol cells were detected by Western blot. Proliferating cell nuclear antigen (PCNA) was selected as nucleoprotein internal control. The quantitative results were shown in Panel (E). (D) Immunofluorescence staining of FOXO3a in A549/Taxol cells was carried out to evaluate the effect of DHW-221 on FOXO3a nuclear translocation. Scale bar = 20 µm. The histograms indicated the percentage of the cells in each condition exhibiting FOXO3a nuclear mean fluorescence intensity (positive cells, green fluorescence) by ImageJ. (F) FOXO3a degradation in A549/Taxol cells with or without DHW-221 co-treatment in different time spot when protein biosynthesis was blocked with 20 µM cycloheximide (CHX). FOXO3a stability was analyzed relative to control by ImageJ software. (G) FOXO3a proteins levels in the presence of MG132 (0.20 μM) or 2.40 μM DHW-221-treated A549/Taxol cells at 24 h. Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 3). Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.