Fig. 3. 5-FU activates MKK3 in CRC but not in primary colonocytes.

a Relative cell number of sh/scr and sh/MKK3 CRCs sublines after 5-FU IC50 treatments as described in “Materials and methods”. Results are reported as mean and S.D. of three independent experiments, and effects in sh/MKK3 + 5-FU condition normalized to relative controls (sh/scr + 5-FU) set to 1.0. Significance was analyzed using unpaired t-test. b Left panel: Western blot on protein lysates of pCDNA3 and pCDNA3-HA-MKK3 transiently transfected Colo205 cells. Blots were incubated with indicated antibodies. More relevant bands from the same filter at same exposure length are reported. Right panel: Relative live cell number in pCDNA3 and pCDNA3 HA-MKK3 cells untreated or 5-FU treated. Data are reported as mean and S.D. of two independent experiments, where HA-MKK3 + 5-FU condition were normalized to those of relative control pCDNA3 + 5-FU set to 1.0. Significance was analyzed using unpaired t-test. c Western Blot analyses were performed on protein lysates derived from cell lines exposed or not to 5-FU. Blots were incubated with indicated antibodies. Representative results of three independent experiments are reported; Densitometry was performed with ImageJ software and relative band intensity was normalized to β-actin and quantified with respect to untreated controls set to 1.0; more relevant bands from the same filter at same exposure length are reported. *p < 0.05, **p < 0.01, ****p < 0.0001, n.s.: not significant