Establishment of inducible shRNA expression in MM cells.

(A) ERK2 levels in JJN-3 cells stably transfected with the tet-repressor protein (pT2-CAG-puro-tTR) and an inducible shRNA cassette against ERK2 (pT2-neo-S2-shERK2) without (lane 3) and with 3 days of doxycyclin treatment (lane 4). Normal wildtype JJN-3 cells in culture (lane 1, basal ERK2 level) and after stable transfection with the pT2-neo-S2-shERK2 expression cassette (lane 2, ERK2 level in constitutive knockdown) serve as references. (B) Concentration- and time-dependence of doxycyclin-induced ERK2 knockdown in pT2-CAG-puro-tTR + pT2-neo-S2-shERK2 JJN-3 cells. Cells were treated with doxycyclin for 24 h. (C) Longer-term time course of induced ERK2 knockdown and ERK2 replenishment after 24 h of treatment of pT2-CAG-puro-tTR + pT2-neo-S2-shERK2 JJN-3 cells with either 1 or 2 μg/ml doxycyclin and subsequent culture for the indicated times. (D) Inducible ERK2 knockdown in different MM cell lines stably transfected with the pT2-CAG-puro-tTR + pT2-neo-S2-shERK2 combination after 3 days of treatment with 1μg/ml doxycyclin.