Fig 1.
TAM and EGFR family receptor profiles of melanoma cell lines.
VGP and MGP melanoma cells were lysed in RIPA buffer and soluble proteins were separated by SDS-PAGE for immunoblotting against indicated antibodies. Shown are representative blots of three experiments.
Fig 2.
Gas6-mediated activation of Axl in melanoma cells.
(A) Indicated melanoma cell lines were transiently transfected with control or Axl siRNA for 2 days prior to starving in quiescence media for 16h. Quiescent cells were then stimulated with indicated factors at the concentrations of 10 nM (EGF) and 200 ng/ml (Gas6) for 10 min followed by lysis in RIPA buffer for immunoblotting using the indicated antibodies. (B) Immunoblotting of indicated proteins extracted from quiescent melanoma cell lines treated with or without 200 ng/ml Gas6 for 10 min. NT: non treatment. Shown are representative blots of three experiments.
Fig 3.
Gas6 promotes migration and invasion of melanoma cells in vitro.
(A) Immunoblotting of indicated proteins extracted from WM852 cells quiesced in quiescent media containing indicated percentage of dialyzed FBS and stimulated with 300 ng/ml Gas6 for 15min. (B) WM852 cells were quiesced in 0.5% FBS quiescent media for 16h and then stimulated with indicated concentration of Gas-6 for 15min. Immunoblottings were performed using indicated antibodies. Shown are representative of three experiments. (C) Quantitative results of relative migratory speed of indicated melanoma cell lines stimulated with indicated factors for 24h. (C) Quantitative results of invaded IgR3 and WM852 with or without an attraction of 500 ng/ml Gas6 for 48h. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, NS: not significant.
Fig 4.
Transient silencing of Axl using siRNA reduces migration and invasion of melanoma cells.
IgR3 and WM852 cells were transiently transfected with Axl siRNA for 48h. (A) Immunoblottings were performed using indicated antibodies to show selective downregulation of Axl. (B) Quantitative results of relative migratory speed of transfected cells with or without 1 μM of R428 treatment. (C) Representative images of DAPI-stained cells invaded through MatriGel and retained on the bottom of transwell membrane. (D) Quantitative results of invaded cells. Shown are representative blots and pictographs of three experiments. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, NS: not significant.
Fig 5.
Stable knockdown of Axl limits migration and invasion of IgR3 cells.
(A) Immunoblottings of stable anti-Axl shRNA expressing IgR3 monoclonal clones using indicated antibodies show selective downregulation of Axl. (B) Quantitative results of relative migratory speed of scramble and Axl shRNA clones. (C)Representative images of DAPI-stained cells invaded through MatriGel and retained on the bottom of transwell membrane. (D) Quantitative results of invaded cells. Shown are representative blots and pictographs of three experiments. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, ** P< 0.01, NS: not significant.