Effect of impairment of p38 function on mitogen-induced DNA-synthesis. (A) Serum-starved NIH3T3 cells were either not pretreated or preincubated for 1 h with SB203580 (10 μM). The cells were then stimulated with 10% FCS, PDGFβ or bFGF (each 50 ng/ml) for 20 h and labelled with [3H]thymidine for additional 4 h in the absence or presence of SB203580. Incorporated radioactivity was determined by liquid scintillation spectrometry. Results are given as x-fold induction of [3H]thymidine incorporation compared to unstimulated, serum-starved cells and are the average ± Sx of a number of four for each run of treatment. The results represent one out of three independent experiments each leading to similar results. (B) NIH3T3 cells were transiently transfected with siRNA directed against p38α or with control siRNA. Knock-down is shown by Western blot analysis. Cells were serum-starved and then stimulated with bFGF or PDGFβ. DNA-synthesis was determined as described in (A). (C) 0.06 × 106 NIH3T3 cells were seeded in 4.5 cm2 dishes. Cells were either not treated or treated with SB203580 (10 μM) 4 h after seeding and then cultured in the absence or presence of SB203580 for another 68 h. Cells were counted in a hemocytometer. The results are expressed as the average ± Sx of a number of four for each time point and treatment. The results represent one out of two independent experiments.