Figure 3
Figure 3. Angiogenic properties in conditioned media from TM4SF5-expressing cells. SNU449 cell lines were seeded in complete RPMI 1640 media containing 10% FBS, rinsed, and incubated in M199 media with 5% FBS (control media) for 48 hours, before collection of conditioned media for HUVECs. The media was changed to a M119 media system, where HUVECs were prepared and maintained. (A) HUVECs were serum-starved for 8 hours, trypsinized, collected, and counted. A total of 5 × 104 cells per well (of 12-well plate) were resuspended with SNU449 cell line–derived conditioned media and seeded in triplicates. The total numbers of cells were counted every 24 hours, and relative cell viability was plotted compared with cells that received control media. (B,C) A total of 105 HUVECs per well (of 24-well plate) were resuspended in conditioned media from SNU449 (P), SNU449Cp (Cp), SNU449Tp (Tp), or SNU449T16 (T16) cell cultures and seeded on Matrigel-coated plates. In case, normal IgG or anti-VEGF antibody (40 μg/mL) was added to the plates (C). Images of capillary-like networks were recorded from 5 randomly isolated fields using a phase-contrast microscope at indicated times (original magnification × 40). After 16 hours of incubation, tube-like structures started to collapse in cultures treated with conditioned media from TM4SF5-null cells, whereas structural integrity was maintained in cultures treated with conditioned media prepared from TM4SF5-expressing cells. (D) Aortic ring segments were prepared and embedded into 2 layers of Matrigel. Normal IgG or anti-VEGF antibody (40 μg/mL) was added to conditioned media from SNU449Tp for 20 minutes. After incubation for 96 hours with conditioned media collected from either SNU449Cp (Cp) or SNU449Tp (Tp) cell cultures, images of endothelial cell outgrowth forming branched cords from the vessel segment margins were obtained using a phase-contrast microscope (original magnification × 40). P values less than .05 were considered significant: *P < .05; **P > .05. Data shown are representative of 3 different experiments.

Angiogenic properties in conditioned media from TM4SF5-expressing cells. SNU449 cell lines were seeded in complete RPMI 1640 media containing 10% FBS, rinsed, and incubated in M199 media with 5% FBS (control media) for 48 hours, before collection of conditioned media for HUVECs. The media was changed to a M119 media system, where HUVECs were prepared and maintained. (A) HUVECs were serum-starved for 8 hours, trypsinized, collected, and counted. A total of 5 × 104 cells per well (of 12-well plate) were resuspended with SNU449 cell line–derived conditioned media and seeded in triplicates. The total numbers of cells were counted every 24 hours, and relative cell viability was plotted compared with cells that received control media. (B,C) A total of 105 HUVECs per well (of 24-well plate) were resuspended in conditioned media from SNU449 (P), SNU449Cp (Cp), SNU449Tp (Tp), or SNU449T16 (T16) cell cultures and seeded on Matrigel-coated plates. In case, normal IgG or anti-VEGF antibody (40 μg/mL) was added to the plates (C). Images of capillary-like networks were recorded from 5 randomly isolated fields using a phase-contrast microscope at indicated times (original magnification × 40). After 16 hours of incubation, tube-like structures started to collapse in cultures treated with conditioned media from TM4SF5-null cells, whereas structural integrity was maintained in cultures treated with conditioned media prepared from TM4SF5-expressing cells. (D) Aortic ring segments were prepared and embedded into 2 layers of Matrigel. Normal IgG or anti-VEGF antibody (40 μg/mL) was added to conditioned media from SNU449Tp for 20 minutes. After incubation for 96 hours with conditioned media collected from either SNU449Cp (Cp) or SNU449Tp (Tp) cell cultures, images of endothelial cell outgrowth forming branched cords from the vessel segment margins were obtained using a phase-contrast microscope (original magnification × 40). P values less than .05 were considered significant: *P < .05; **P > .05. Data shown are representative of 3 different experiments.

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