Fig. 4. TNF- stimulates anchorage-dependent and -independent growth of 2A4 cells. (A,B) 35 mm tissue culture dishes were seeded with 2A4 cells at a density of 2x10⁵ cells per dish in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was renewed and the cultures were left untreated or were treated with 10 ng ml^-1 TNF-. 5 days later, control cells have formed only small colonies (A), whereas TNF--treated cells have formed a nearly confluent monolayer (B). Scale bars, 200 µm. (C) Dose dependence of TNF--induced cell proliferation. 2A4 cells were seeded in 16 mm wells at 1x10⁴ cells per well in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was removed, fresh medium was added, and the cultures were incubated with or without various concentrations of TNF-. Medium and treatments were renewed every 2-3 days. Cells in triplicate wells were harvested by trypsinization after 9 days and counted with a haemocytometer. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. ^*P<0.0005 (compared with control values). (D) Time course of TNF--induced cell proliferation. 2A4 cells were seeded in 16 mm wells at a concentration of 1x10⁴ cells per well in serum-free DMEM-F12 medium supplemented with ITS+ Premix. After 24 hours, the medium was changed and the cells were incubated for 4 days, 8 days, 12 days and 16 days with or without 10 ng ml^-1 TNF-. Cells in triplicate wells were harvested and counted at the indicated time points. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. ^*P<0.0005 (compared to controls at 4 days). (E,F) Assay of anchorage-independent growth. 2A4 cells were suspended at 3x10⁴ cells ml^-1 in agarose gels and grown for 14 days in the absence (E) or presence (F) of TNF-. Scale bar, 200 µm. (G) Quantification of colony formation. Three randomly selected fields per experimental condition were photographed, and the number of colonies greater than 40 µm was determined in each photographic field. Data represent the means ± s.e.m. of at least three independent experiments. Mean values were compared using Student's unpaired t test. ^*P<0.025; ^**P<0.001 (compared with control values).