Figure 5

Overexpression of Lon enhances cell proliferation via ROS-dependent Ras-ERK1/2 signaling. (a) Overexpression of Lon activates ROS-dependent MAPK and Ras-c-Raf-MEK1/2-ERK1/2 signaling. Ectopic expression of Lon in 293 T cells is performed by transfected with pcDNA3-Lon. The expression of Lon, phosphorylated signaling kinases were detected by western blotting using indicated specific antibodies. The ROS production was inhibited by addition of NAC (1, 5, or 10 mM) for 24 h. Result is presented as a representative of three independent experiments. (b) Downregulation of Lon diminishes the MAPK activation triggered by increased Lon FADU cells were retrovirally infected with pMKO vector, Lon-pBABE-puro, or pMKO expressing shRNAs for human Lon. Immunoblots were probed with indicated antibodies and anti-GAPDH antibody acts as a loading control. Result is presented as a representative of three independent experiments. (c and d) Overexpression of Lon enhances cell proliferation via ERK signaling. 293T cells overexpressing Lon were treated with 20 μM PD98059 (PD, MEK inhibitor), 5 μM SB203580 (SB, p38 inhibitor), and 10 μM SP600125 (SP, JNK inhibitor) for 24 h. The protein levels of cyclin A, D1, and E were analyzed by western blotting using indicated antibodies (c) Result is presented as a representative of three independent experiments. For cell growth curves, 293T cells overexpressing Lon were seeded onto 24-well plates (2 × 10⁵ cells/well). After 20 h, the cells were left non-treated or treated with the inhibitors and the cell proliferation was measured using the MTT assay at the indicated time intervals (d)