TWIST expression in HS5 stroma and effect on apoptosis in cocultured KG1a cells. (A) Expression of TWIST in HS5 cells transfected with scrambled siRNA (SCR) or TWIST-specific siRNA (KD TWIST 1.1 and KD TWIST 1.2). TWIST expression was determined by Western blot. β-Actin served as control. (B) Western blot of PYCARD protein in KG1a cells in contact cultures with HS5 cells, transfected with either a scrambled sequence (SCR) or with a TWIST-specific siRNA (KD TWIST), respectively. β-Actin served as loading control. PYCARD served as additional protein control for the stroma effect because we had shown previously up-regulation of PYACRD in KG1a cells after stroma contact.¹²  (C) Earl-stage apoptosis induced by TNFα (25 ng/mL) in KG1a cells that remained stroma-attached in control cultures containing unmodified HS5 cells (HS5 wt) and in cultures with HS5 cells transfected with either a scrambled siRNA sequence (HS5 scr) or siRNA specific for TWIST (HS5 KD TWIST). Apoptosis was determined by flow cytometry. Only CD45⁺ (KG1a) cells were considered. KG1a cells that had detached from stroma are not included. (D) Early-stage apoptosis induced by TNFα (25 ng/mL) in KG1a cells in contact with HS27a or normal bone marrow stroma (NBM), either unmodified (wt) or modified by transfection with a scrambled siRNA sequence (scr) or siRNA specific for TWIST (KD TWIST). Apoptosis was determined by flow cytometry. Only CD45⁺ (KG1a) cells were considered. KG1a cells that had detached from stroma are not included. Error bars indicate SEM.