Impact of MEK1 or MEK2 silencing on the proliferation of human colon carcinoma cell lines. (A) Immunoblot analysis of MEK1, MEK2 and phospho-MEK1/2 expression in the human colon carcinoma cell lines HCT116, HT-29 and SW480, and in the breast adenocarcinoma line MDA-MB-231. Lysates were prepared from cells deprived of serum for 24 h (c), restimulated with serum for 5 min (+), or proliferating exponentially (exp). (B to D) HCT116 (B), SW480 (C) and HT-29 (D) cells were infected with MEK1 or MEK2 shRNA-encoding lentiviruses. A non-silencing inactive MEK1 shRNA (NS) was used as negative control. The cells were replated 72 h (day 0) after infection to monitor the expression of MEK1 and MEK2 and to measure the rate of cell proliferation. Expression of MEK isoforms was analyzed by immunoblotting at day (D) 1, 3 and 5 (left). Cell proliferation was measured each day using the MTT assay (right). For experiments with small-molecule MEK1/2 inhibitor, the cells were treated with 10 μM U0126 added at 48 h intervals.