Figure 8. Real-time PCR and Western blotting showing the expression of LC3, Beclin-1 and FOXO3a in hFOB1.19 osteoblasts. Cells were pretreated with or without SRT1720 and EX527 for 2 h, and then cultured with 17β-E2 (10-6 M) for 24 h. RT-PCR and Western blot assay showed the expression of LC3 (A, B), Beclin-1 (C, D) and FOXO3a (E, F) mRNA and proteins in osteoblasts. The mRNA data were normalized to GAPDH. The histograms showed the relative proteins expression of LC3, Beclin-1 and FOXO3a normalized to β-actin, and the densitometry analysis of the blots were estimated by using the Image J Software. The data were expressed as the means ± SD from 3 independent experiments. Statistically significance was evaluated using the Student±s t-test. * P < 0.05 vs. the control group was considered significant.