Expression of Ikaros proteins in HMCLs and induction of cytotoxicity after knockdown of IKZF1 and IKZF3. (A) Expression of IKZF1 and IKZF3 in HMCLs. Nine HMLCs either sensitive or resistant to lenalidomide were analyzed for the expression of Ikaros by western blot. (B) Expression of IKZF1 in HMCLs was detected by flow cytometry analysis. (C-D) Knockdown of IKZF1 and IKZF3 induced cytotoxicity. MM1.S cells were infected with lentivirus-expressing nontargeting control or IKZF1 or IKZF3 shRNAs. MTT assays were performed at day 6 after infection and western blot analysis was performed at day 3 after infection. (E-G) Overexpression of IKZF1 and IKZF3 in a lenalidomide-sensitive cell line, MM1.S, did not substantially change lenalidomide sensitivity. MM1.S cells were infected with control virus (expressing vector alone) or lentivirus-expressing IKZF1 and IKZF3 after 72 hours (E), or after sorting GFP⁺ cells in MM1.S cells infected by IKZF1 lentivirus at 14 days after infection (F), cells were seeded in a 96-well plate and were treated with lenalidomide. (G) MTT assay was performed at day 6 after treatment and western blot analysis was performed at day 3 to confirm overexpression of IKZF1 and IKZF3.