Fig 2. Establishment and characterization of vectors for concatenated shRNA expression cassettes.

(A) Western blot showing the effects of selective ERK1 and/or ERK2 knockdown in JJN-3 MM cells. Cells were transfected with plasmid solutions by electroporation, and subsequently purified based on the presence of CD4Δ (see Methods and [8]). Cells were then harvested for Western blotting at day three post-transfection. Knockdown of both ERK1 and ERK2 from the pSUS double-cassette vectors (lanes 2 and 3) matches the effect obtained with the pSUPER-based single-cassette vectors (lanes 4–6). (B) Left: Establishment of knockdown constructs for MEK2 (three different target sequences tried), based on lowered expression of HA-tagged human MEK2 from a co-transfected expression plasmid (see Methods). The shMEK2-2 sequence was chosen for further experiments (= shMEK2). Right: Knockdown of MEK1 and MEK2 from a pSUS double cassette vector (middle lane) versus combined transfection of the single shRNA expression vectors (right lane). (C) Concentration-dependent effects of the MEK1&2 inhibitor PD0325906 on the levels of phospho-ERK1/2 and phospho-MEK1/2. Cells were treated for 1 h with the drug and harvested for Western blotting. Substantial downregulation of phospho-ERK1/2 results in inceased levels of phospho-MEK1/2. FACS analysis with staining for early (annexin V) and late (PI) apoptotic markers after 3 days of drug treatment does not show increased rates of apoptosis. (D) Western analysis of the efficiency of MEK1+2 and ERK1+2 knockdown from a quadruple cassette shRNA-expression vector (lane 6) in relation to single cassette vectors (ERK1, ERK2, lanes 2 and 3) and double cassette vectors (ERK1+2, lane 4; MEK1+2, lane 5). Cells were harvested at day 3 post-transfection.