Figure 6 | Cell Death & Disease

Figure 6

From: Overexpression of Lon contributes to survival and aggressive phenotype of cancer cells through mitochondrial complex I-mediated generation of reactive oxygen species

Figure 6

Overexpression of Lon induces EMT through ROS-dependent MAPK signaling. (a) Lon expression is upregulated in metastatic prostate tumor cells. Box plots showed relative Lon mRNA level of benign, primary, and metastatic tumor cells in the public data sets GDS1439. (b) Upregulation of Lon increases the protein level of MMP-2. OEC-M1 or FADU cells were stably transfected with plasmid encoding Lon. The cell extracts were immunoblotted with indicated antibodies and antibody to GAPDH as a loading control. (c) Overexpression of Lon induces cell migration. 293 and 293/Lon cells were seeded in Oris 96-well, and removed the stopper after 16 h. Cell migration was analyzed by their motility ability after 24 and 48 h. The diameter of cell-free area was measured (Supplementary Figure S8), and data were collected and quantitated from three independent experiments. **P<0.01. (d) Downregulation of Lon reduces cell migration. FADU or SCC-15 cells were retrovirally infected with pMKO vector, Lon-pBABE-puro, and/or pMKO expressing shRNAs for human Lon. Cells were seeded in Oris 96-well, and removed the stopper after 16 h. Cell migration was analyzed by their motility ability after 24 and 48 h. The diameter of cell-free area was measured as described in Figure 6c, and data were collected and quantitated from three independent experiments. *P<0.05; **P<0.01. (e) EMT-like cell morphology was observed in 293 cells overexpressing Lon (293/Lon). Scale bar was shown. (f) Overexpression of Lon induces ROS-dependent EMT. Ectopic expression of Lon in 293T cells is performed by transfected with pcDNA3-Lon. The protein expression was detected by western blotting using the indicated 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. (g) Overexpression of Lon induces EMT via ERK and p38 signaling. 293T cells overexpressing Lon were treated with 20 μM PD98059 (PD, MEK inhibitor), 5 μM SB203580 (SB, p38 inhibitor) and 10 μM LY294002 (LY, PI3K inhibitor) for 24 h. The protein levels were analyzed by western blotting using indicated antibodies. The result is a representative of those obtained in at least three individual experiments

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