Figure 1.

(a) CD138 expression on 5T33 MM cells analyzed by flow cytometry. Purified 5T33 MM cells were analyzed by a membranic staining with PE labeled anti-CD138 and simultaneous staining with anti-5T33 MM idiotype antibodies, which were detected with rat anti-mouse IgG1-APC. Irrelevant isotype controls were used as control. Dot plot represents anti-idiotype and anti-CD138 staining of events gated on 7AAD- living cells. (b, c) B-cell lymphoma 6 (Bcl-6), B-lymphocyte-induced maturation protein 1 (Blimp-1), X-box binding protein-1 (Xbp-1) and Interferon regulatory factor 4 (IRF4) expression in 5T33 MM and 5TGM1 sorted CD138− cells compared with CD138+ cells. Real-time Q-PCR showing fold increase/decrease of mRNA levels in CD138− samples compared with CD138+ samples (equal to 1). To standardize the amount of sample RNA, we used an endogenous reference gene. Results of one independent sample of three are shown. Primer sequences are available upon request. (d) Expression of EZH2, SUZ12 and EED in the 5T33 MM CD138− cells compared with CD138+ cells. Real-time Q-PCR showing fold increase/decrease of mRNA levels in CD138− samples compared with CD138+ samples (equal to 1). To standardize the amount of sample RNA, we used an endogenous reference gene. Results shown are combined from two independent samples repeated twice. Primer sequences are available upon request. (e) Clonogenicity of CD138− and CD138+ populations in 5T33 MM model. Sorted populations were plated in methylcellulose medium at a concentration of 5×10⁴/ml. After 10 days incubation at 37 °C and 5% CO₂, the number of colonies was counted. The mean value±s.d. of two (5T33 MM) and three (5TGM1) independent experiments is shown.