MLN2238 oroverexpression ofmiR33b negatively regulates PIM-1 signaling. (A-B) MM.1S cells were transiently transfected with either pre-miR33b or scrambled probe using the Cell Line Nucleofector Kit V, and cells were harvested 24h after transfection, followed by analysis of PIM-1 transcripts (A) and PIM-1 protein levels (B) by qRT-PCR and Western blot analysis, respectively. (C-D) MM.1S cells were treated with MLN2238 (12nM) for the indicated times, and cells were harvested. Total RNA and protein extracts were subjected to analysis of PIM-1 transcripts (C) and PIM-1 protein levels (D) using qRT-PCR and Western blotting, respectively. The relative expression of PIM1 to β-actin was quantified using ImageJ Version 1.38x software. (E) MM.1S cells were transfected with either scramble siRNA or PIM-1 siRNA. Total protein lysates were subjected to immunoblot analysis using Abs specific against PIM-1, phospho-Bad, and actin. (F) MM.1S cells overexpressing miR33b or MLN2238-treated MM.1S cells were harvested and protein lysates were subjected to immunoblot analysis using Abs specific against phospho-Bad and actin. (G) MM.1S cells stably expressing pWzl-neo retroviral vector or PIM1 (Addgene) were established by retrovirus infection. The stable cell lines were then transfected with miR33b or treatment with MLN9708 (12nM) for 24 hours. The cell viability was measured with the MTT assay. Results are shown as means ± SD (n = 3).