Fig. 3. LZ-101 induces mitochondrial apoptosis by up-regulating FOXO3a expression in A549 cells.

a The levels of FOXO3a and Bim were assessed by Western blot after 5, 10, and 15 μM LZ-101 treatment for 24 h. b Bim mRNA was measured by real-time PCR following treatment with 5, 10, and 15 μM LZ-101 for 24 h in A549 cells. β-actin was used as internal control. The relative levels were calculated to β-actin mRNA expression. c The transcriptional activities of FOXO3a in A549 cells co-transfected with pGMFOXO-Lu and pRL-TK Renilla with LZ-101. Luciferase activity was determined 24 h post-treatment by promega dual luciferase reporter assay system, normalized against values for the corresponding pRL-TK Renilla activity. d Immunofluorescence staining of FOXO3a in A549 cells (treated with 15 μM LZ-101) was carried out to test the effect of LZ-101 on FOXO3a protein nuclear translocation. Scale bars, 50 µm (left). Histograms shows the fold change of nuclear FOXO3a positive cells for three independent experiments (mean ± SD) (right). FOXO3a siRNA was transfected into A549 cells. The cells were cultured in serum free medium overnight. After 8 h, cells were treated with 15 μM LZ-101 for 24 h. e FOXO3a, Bim, Bax, and Bcl-2 were detected by Western blot (top). Ratio of Bax/Bcl-2 expression using densitometric analysis (bottom). f Annexin-V/PI staining assay and g the change of ΔΨ_m were measured by flow cytometry. Data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01 compared with DMSO group; ^#P < 0.05, ^##P < 0.01 compared with LZ-101 treatment group