Figure 3.

FOXO3a is downregulated in cisplatin-resistant osteosarcoma cells. Heat maps of the 30 most significantly (P < 0.01) altered genes in cisplatin-resistant osteosarcoma cells were shown. (A) The RNA from MG63, MG63-R12, U2OS and U2OS-R5 cells was subjected to miRNA microarray analysis. Each column represents a tissue sample, and each row represents a probe set. The heat maps indicate high (red) or low (green) levels of gene expression. (B) qRT-PCR was performed to verify the expression of Hsp110, ATG5, ATG7, Beclin-1, PUMA, Caspase-3, Bcl-6 and FOXO3a in cisplatin-resistant osteosarcoma cells. The relative expression of these genes in MG63-R12 and U2OS-R5 cells was normalized to their corresponding parental cells (P < 0.001). (C) The FOXO3a protein level was downregulated in cisplatin-resistant osteosarcoma cells. MG63, MG63-R12, U2OS and U2OS-R5 cells were plated in 96-well plates containing 0.1 ml of 0.5% FBS medium for 24 hrs. The cells were collected and subjected to Western blot analyses for FOXO3a or β-Actin. (D) The activation of autophagy resulted in the downregulation of FOXO3a. MG63, MG63-R12, U2OS and U2OS-R5 cells were plated in 96-well plates. After 24 hrs, the culture medium was replaced with 0.1 ml of fresh medium containing 0.5% FBS and the same medium containing 500 nM rapamycin for another 24 hrs. The cells were collected and subjected to Western blot analyses for LC3, FOXO3a or β-actin. (E and F) The inhibition of autophagy resulted in the accumulation of FOXO3a. MG63, MG63-R12, U2OS and U2OS-R5 cells were treated with or without 3-MA (E), and the MG63-R12 and U2OS-R5 cells were transfected with lentivirus containing either ConshRNA or ATG5-shRNA (F). The cells were collected and subjected to Western blot analyses for FOXO3a, LC3, ATG5 or β-actin. The data represent three independent experiments.