Inhibition of TWIST and ICAM1 (CD54) expression in HS5 cells. (A) FACS analysis of ICAM1 expression in HS5 cells transfected with scrambled siRNA (SCR) or TWIST-specific siRNA (KD TWIST). The cells were stained with FITC conjugated anti–human CD54 antibody (AB) for 30 minutes (a indicates isotype control; b, HS5 transfected with scrambled siRNA sequences; c, HS5 transfected with TWIST-specific siRNA.) (B) Western blot of TWIST and ICAM1 proteins in HS5 cells transfected with scrambled siRNA (SCR) or with 1 of 3 TWIST-specific siRNAs (KD TWIST in HS5). β-Actin served as loading control. (C) IACM1 expression in primary MDS stroma cells; a indicates normal bone marrow stroma labeled with isotype control antibody; b, normal bone marrow stroma labeled with CD54+-allophycocyanin flow antibody; c1, patients with RA MDS; c2, patients with RAEB-2 MDS. (D) Early stage apoptosis (annexin V+/propidium iodide−) in KG1a cells in control cultures containing unmodified HS5 cells (WT) or HS5 cells pretreated with either 5 or 10 μg of anti-ICAM1 antibody. Cells were cultured in the absence or presence of TNFα (25 ng/mL). Apoptosis was determined by flow cytometry. Only CD45+ (KG1a) cells were considered. (E) Early-stage apoptosis in PL-21 and KG1a cells cultured with either unmodified stroma or stroma pretreated with anti-IACM1 antibody, and apoptosis in PL-21 or KG1a cells treated with anti-CD11b antibody. Error bars indicate SEM.
