Effect of rictor knockdown in MM1.S and 8226 MM cell lines. (A) and (D) MM1.S and 8226 cells were infected with lentivirus expressing shRNA targeting rictor (rictor) or control shRNA containing a nontargeting sequence (scr). Cells were infected at MOIs of 20:1 or 50:1. Infected MM1.S cells were assayed by immunoblot 3 days after infection for expression of rictor, actin, phospho-AKT, total AKT, phosphorylated NDRG1 (on serine 330), or total NDRG1. Infected 8226 cells were selected in puromycin and stably expressing lines were similarly assayed by immunoblot for rictor, actin, phospho-NDRG1 and total NDRG1. (B-C) MM1.S cells infected with lentivirus expressing rictor-targeted shRNA (shRNA) or control shRNA (scr) at 20:1 or 50:1 MOIs were assayed 5 days after infection for number of viable cell recovery (B) or for percent apoptosis (C) where apoptosis was assayed by flow cytometric analysis of activated caspase 3. The experiment in (B) and (C) was repeated once with identical results. (E) Stable 8226 cell lines expressing rictor shRNA after 20 or 50:1 MOI infections and in vitro selection (rictor 20, rictor 50) or control (scr 20, scr 50) sequence and in vitro selection, were seeded at 105 cells/mL and assayed for cell growth in vitro over 9 days. Data are presented as fold increase in viable cell numbers on a log scale, mean ± SD (n = 4).
