Figure 6

The inhibition of the RAF/MEK pathway in CLL cells causes downregulation of Mcl-1 protein. (A) CLL cells purified from frozen PBMCs were cultured in the presence of 30 nmol/L CXCL12 and 50 µmol/L PD98059 or the equivalent DMSO control for 17 h and analyzed by Western blot for the expression of Mcl-1 and GAPDH. Western blot data from one representative CLL sample out of four is shown. Densitometry analysis was performed for each patient sample to quantitatively evaluate the levels of Mcl-1 expression, which is depicted as a ratio of Mcl-1 expression over GAPDH, normalized to media control. Data shown are means ± SD from four different CLL samples. *Statistically significant difference: P < 0.05 (paired Student t test). (B) CLL cells purified from frozen PBMCs were cocultured with MSCs for 18 h in the presence of 10 µmol/L sorafenib, 50 µmol/L PD98059, 10 µmol/L GW5074 or DMSO as control. Viability was assessed by flow cytometry using PI-DiOC6 staining. Data shown are mean ± SD for three patients. *Statistically significant difference: P < 0.05 (paired Student t test). At the same time as viability was assessed, cells were collected and lysed for Western blot analysis, as described in Materials and Methods. Representative results of the Mcl-1 and β-actin Western blot are shown for one out of three patients.