Effect of lenalidomide and thalidomide on mRNA of ABCG2 transcript, clonogenic potential, and signaling pathways of SP cells. (A) Quantification of the relative mRNA expression of ABCG2 and (B) ABCB1 (MDR/Pgp) in SP or MP cells treated with lenalidomide and thalidomide (10μM) for 6 hours was assessed using QuantiGene Plex assay. Fluorescence intensity of ABC transporters was normalized to fluorescence intensity of the housekeeping gene GAPDH. All experiments were performed in triplicate, P < .05, t test, statistical significance. (C) Sorted SP cells, with or without treatment with lenalidomide (LEN; 1μM) and thalidomide (THAL, 1μM) for 72 hours, were restained with Hoechst 33342. (D) Clonogenic assay of sorted SP and MP cells treated with lenalidomide and thalidomide (10μM) is shown at day 14. (E) Images of colonies derived from SP cells, both control and treated with lenalidomide and thalidomide, were obtained using a Leica inverted microscope (with a Leica DFC300 Fx camera [4×/0.1, 10×/0.22, and 20×/0.35] Leica IM50 image-acquisition software Version 4). (F) Multiplex analysis of lenalidomide-induced changes in phosphorylation of signaling pathways in sorted SP and MP fractions or control OPM1 cells. The different populations of cells were treated with lenalidomide (1 or 10μM) for 1 hour. Protein concentrations of whole-cell lysates were measured using a Bradford protein assay kit and normalized to a panel of 5 total proteins. A multiplex panel of 16 phosphoproteins (Akt, c-Jun, ERK1/2, GSK-3α/β, HSP27, IRS-1, JNK, MEK1, NF-κB p65, p38 MAPK, p53, p70 S6 kinase, p90RSK, Src, STAT3, STAT6) was analyzed in a 96-well format using the Bio-Plex suspension array system. Relative amounts of phosphorylation in treated vs control untreated cells are shown, and fold changes in expression are depicted in a color-coded scale.