Effect of lenalidomide and thalidomide on viability, proliferation, and stemness markers (SP cell fraction and CD138^−/low+ population) of OPM1 in the absence and presence of BMSCs. (A) Cytometric dot plots represent CFSE-labeled OPM1 cells alone (first 2 dot plots) or in coculture with unlabeled-HS-5 BMSCs (last 2 dot plots), showing CFSE profiles together with 7-AAD⁺ cells, and percentage of SP cells by Hoechst 33342. Analysis gated only on CFSE⁺/7-AAD⁻ OPM1 cells at 1 day. (B) Percentage of SP fraction in control and lenalidomide and thalidomide treated OPM1 cells, both alone and with BMSCs, decreased in a dose-dependent manner at 3 days. (C) Fluorescence intensity of CFSE⁺ 7-AAD⁻–labeled OPM1 cells, both alone or with HS-5 BMSCs, is shown after treatment with lenalidomide and thalidomide (0.1, 1, 5, and 10μM). (D) Percentage of CD138^−/low+ OPM1 cells at 3 days is shown in control cultures; as well as after treatment with lenalidomide and thalidomide alone, or in coculture with BMSCs. (E) Viability of CFSE-labeled OPM1 cells at 3 days decreased after drug treatment in a dose-dependent manner (0.1, 1, 5, and 10μM), assessed using 7-AAD staining and a FACSAria Special Sorter UV laser flow cytometer.