![Figure 2. Ectopic expression of TM4SF5 in SNU449 cells enhanced VEGF expression and secretion. (A) Parental SNU449 cells were transfected with pGL3-human VEGF promoter and pBabe–β-galactosidase together with a mock construct, pcDNA3-TM4SF5, or Myc-(His)6-TM4SF5. After 2 days, lysates were prepared and luminescence was measured. Transfection efficiency was normalized by β-galactosidase activity. Data are mean plus or minus SD of 3 independent experiments. RLU indicates relative luciferase activity units. (B,F) TM4SF5-null SNU449 cells were transiently transfected with either the mock construct or pcDNA3-TM4SF5. One day later, whole-cell lysates were prepared, normalized, and analyzed by standard Western blots for the indicated proteins. (C) Whole-cell extracts prepared from parental SNU449 (P), control SNU449Cp (Cp), or stably TM4SF5-expressing SNU449 cell lines (Tp or T16) were analyzed for VEGF expression using 2 different anti-VEGF antibodies (rabbit anti-VEGF polyclonal and mouse anti–VEGF-A [c-1] monoclonal antibody from Santa Cruz Biotechnology, B) or the indicated molecules (F). (D) Subconfluent SNU449Cp and SNU449Tp cells were rinsed and incubated with serum-free media for 24 hours before collection of conditioned media. Using conditioned media from each cell line, angiogenic factors were analyzed using RayBiotech human angiogenesis antibody array. Notably, only VEGF was elevated in conditioned media from TM4SF5-expressing SNU449Tp cells. (E) One day after SNU449Cp or SNU449Tp cells were seeded, subconfluent cells were treated with dimethyl sulfoxide or various pharmacologic inhibitors (U0126, 20 μM; PD98056, 40 μM; PP2, 40 μM; PP3, 40 μM; AG490, 30 μM; Y27632, 15 μM) for 24 hours. Whole-cell extracts were normalized and used in standard Western blots for the indicated molecules. Notably, c-Src family kinase inhibitor PP2 mostly reduced VEGF expression in SNU449Tp cells. (G-J) TM4SF5-positive SNU449Tp (G-I) or Huh7 (J) cells were transfected with shRNA against GFP (shGFP) or TM4SF5 (shTM4SF5) (G,J), or siRNA against GFP, c-Src (H), or STAT3 (I). After 48 hours, whole-cell lysates were prepared, normalized, and used in standard Western blots for the indicated molecules. Immunoblotting with anti-VEGF antibody (pAb) resulted in multiple bands, presumably isotypes. Data shown were representative from 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/8/10.1182_blood-2008-05-160671/5/zh80070930960002.jpeg?Expires=1735513481&Signature=zMM--XbnAbvFojGmy9LWSw-7boCyN2eBqWh5YCFVTTt-~gNpruiF7VQi--eu1uQMngxDA0huVRWtRFmqDadAiO~hQGN~Gsa0vnipMYfJ7FLyfosmFV~wN6EGNyxM8gbSg1Dj4Qy1u8RkROmvacMRkrHJL7j2C7O~QOW5etOwaUFUfZvN~8HnBS4XLR8KXGV1J4RDO6WXoCe~56XrkbGi9yIc5Ga~sV~QKNuvxFVtDPsPwY1Lr7wgEJbvqk-~EtsBhTiVLU5tVEZ3jIh4a8s5j-BPvD3jYOqhWnZ~uTkJ~EflcDArk9Nz2KbZ6DS34NjS6h5fw8dGSlY~NG0kuHqwag__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ectopic expression of TM4SF5 in SNU449 cells enhanced VEGF expression and secretion. (A) Parental SNU449 cells were transfected with pGL3-human VEGF promoter and pBabe–β-galactosidase together with a mock construct, pcDNA3-TM4SF5, or Myc-(His)6-TM4SF5. After 2 days, lysates were prepared and luminescence was measured. Transfection efficiency was normalized by β-galactosidase activity. Data are mean plus or minus SD of 3 independent experiments. RLU indicates relative luciferase activity units. (B,F) TM4SF5-null SNU449 cells were transiently transfected with either the mock construct or pcDNA3-TM4SF5. One day later, whole-cell lysates were prepared, normalized, and analyzed by standard Western blots for the indicated proteins. (C) Whole-cell extracts prepared from parental SNU449 (P), control SNU449Cp (Cp), or stably TM4SF5-expressing SNU449 cell lines (Tp or T16) were analyzed for VEGF expression using 2 different anti-VEGF antibodies (rabbit anti-VEGF polyclonal and mouse anti–VEGF-A [c-1] monoclonal antibody from Santa Cruz Biotechnology, B) or the indicated molecules (F). (D) Subconfluent SNU449Cp and SNU449Tp cells were rinsed and incubated with serum-free media for 24 hours before collection of conditioned media. Using conditioned media from each cell line, angiogenic factors were analyzed using RayBiotech human angiogenesis antibody array. Notably, only VEGF was elevated in conditioned media from TM4SF5-expressing SNU449Tp cells. (E) One day after SNU449Cp or SNU449Tp cells were seeded, subconfluent cells were treated with dimethyl sulfoxide or various pharmacologic inhibitors (U0126, 20 μM; PD98056, 40 μM; PP2, 40 μM; PP3, 40 μM; AG490, 30 μM; Y27632, 15 μM) for 24 hours. Whole-cell extracts were normalized and used in standard Western blots for the indicated molecules. Notably, c-Src family kinase inhibitor PP2 mostly reduced VEGF expression in SNU449Tp cells. (G-J) TM4SF5-positive SNU449Tp (G-I) or Huh7 (J) cells were transfected with shRNA against GFP (shGFP) or TM4SF5 (shTM4SF5) (G,J), or siRNA against GFP, c-Src (H), or STAT3 (I). After 48 hours, whole-cell lysates were prepared, normalized, and used in standard Western blots for the indicated molecules. Immunoblotting with anti-VEGF antibody (pAb) resulted in multiple bands, presumably isotypes. Data shown were representative from 3 independent experiments.
