AJs sequester Fas at site of cell–cell junction, and this impairs efficient FasL binding and therefore Fas cell death signaling. (A) Binding assays of latex beads coated with FasL (Beads FasL) on HCT15 transfected with siRNAs against α-catenin and followed by phase-contrast microscopy. (B) FasL-coated or -noncoated (NC) beads attached to cells were counted using flow cytometry after cell lysis. Graphs represent means ± SEM (n = 3). (C and D) Detection of Fas and cleaved caspase-8 in HCT15 cells incubated with FasL-coated beads was done by IF at different time points. Beads appear in red (by autofluorescence). An XZ projection of the Z stack is shown. Arrows indicate the accumulation of Fas beneath the beads. (E) Accumulation of Fas and activated caspase-8 under FasL-coated beads was quantified. Graph represents mean ± SEM (n > 3). (F) FasL-coated beads were used to immunoprecipitate activated Fas from cell lysates of HCT15 cells transfected with indicated siRNAs. Fas, caspase-8, and FADD were detected by IB. Arrows and arrowheads indicate native caspase-8 and cleaved (activated) caspase-8, respectively. (G) Relative caspase-8 and Fas were quantified by densitometry at t = 0 or after 30 min FasL-coated bead activation (n = 3). Bars: 10 µM (C and D); 50 µM (A). *, P < 0.05; **, P < 0.01; Student’s t test.