Integrin-β7 expression in myeloma cell lines and primary myeloma cells. (A) Flow cytometric analysis demonstrating integrin-β7 expression in MM cell lines (OPM2, INA6, MM1S, 8226, H929, and U266). Open histograms represent isotype IgG1 control, whereas solid histograms indicate integrin-β7 staining. (B) ITGB7 mRNA expression as determined by quantitative RT-PCR in indicated MM cell lines and CD138⁺ sorted cells from the BM aspirates of MM patients. Data quantification was carried out by the 2^−ΔΔct method relative to a reference human cDNA library (Stratagene). (C) Myeloma tissue microarray (TMA) constructed from the BM biopsies of 79 newly diagnosed MM patients was used to evaluate the expression of integrin-β7 and Cyclin D2 by immunohistochemical staining (IHC) and its impact on prognosis. Shown in the insets are representative H&E, Cyclin D2, and integrin-β7 staining of MM patients BM biopsies. Also shown, positive and negative controls (Cyclin D2: positive OPM2 myeloma cell line; negative: human tonsils; Integrin-β7: positive MM1S myeloma cell line; negative: HL60 leukemia cell line). Images were acquired with a bright light Olympus BX5 microscope and multispectral camera (Nuance Fx; CRi) 10× magnification. Kaplan-Meier survival curves indicate the shorter time to progression (TTP) for patients with MM cells coexpressing Cyclin D2 and integrin-β7. (D) ITGB7 silencing with lentiviral mediated delivery of ITGB7-specific shRNAs (shRNA 2 and 3) in MM1S, H929 and INA-6 cells. Shown is integrin-β7 expression in puromycin-selected cells transfected with ITGB7-specific shRNAs (solid histogram: ITGB7^silenced) or scrambled oligonucleotides sequences (open histogram, dashed line: ITGB7^positive) relative to control IgG1 istoype (open histogram, solid line) as determined by flow cytometry. (E) qRT-PCR confirming ITGB7 silencing in established puromycin-resistant ITGB7^silenced versus ITGB7^positive cells.