TNFR1-mediated signaling in KG1a is required for apoptosis in KG1a cells cocultured with HS5 and exposed to TNF-α. (A) Knockdown of TNFR1 in KG1a cells with constructs nos. 2 and 3, as described in “Methods,” resulted in significant decrease of TNFR1 mRNA expression. (B) The decrease in RNFR1 mRNA in KG1a cells was reflected in reduced cell surface expression of the receptor protein. Shown are results after transfection with TNFR1 construct no. 2 (□) sorted into GFP⁻ cells (not containing the construct) and GFP⁺ (containing the construct). Transfection with a construct for TNFR2 (construct no. 2) did not alter the expression of TNFR1. Receptor expression levels are given relative to cells that were not subjected to the transfection procedure (shown is 1 of 2 identical experiments). (C) Transfection with the same constructs (nos. 2 and 3) that reduced message (A) also decreased apoptosis significantly. (D) In coculture with HS5 cells, nontransfected (GFP⁻) KG1a cells were sensitive to apoptosis induced by both TNF-α (100 ng/mL) and TRAIL (300 ng/mL). In contrast, GFP⁺ (miR-TNFR1) cells were resistant to TNF-α–induced apoptosis, but remained sensitive to TRAIL-induced apoptosis. (E) Stable knockdown of TNFR1 or TNFR2 (4 constructs each) in HS5 cells failed to attenuate stroma dependent TNF-α–induced apoptosis in cocultured unmodified KG1a cells. ψ indicates P < .01; Ω indicates P < .001