Figure 5.

Effect of cholesterol depletion on CYLD-downregulated cells. (A,B) Localization of EGFR, lipid rafts, and CYLD (A) or anti-hemagglutinin (HA) (B) as analysed by immunofluorescence staining. HSC3 cells were transfected with the indicated siRNA and plasmids expressing deletion constructs of CYLD. After incubation for 48 h, cells were starved for 12 h in a serum-free medium. Cells were then stained with the appropriate antibodies and observed under fluorescent microscopy. Scale bars, 10 µm. (C,D) Effects of nystatin on EGF- or cetuximab (CTX)-induced EGFR endocytosis. HSC3 cells were transfected with siRNA and incubated for 48 h, then, after 12 h of incubation in a serum-free medium, cells were pretreated with 25 µg/mL of nystatin for 30 min before stimulation with 100 ng/mL of EGF for the indicated times (C) or 100 µg/mL of CTX for 60 min (D). EGFR localization was analysed via immunofluorescence staining. Scale bars, 20 µm. (E) Effects of nystatin on total EGFR expression after CTX treatment in CYLD-downregulated cells. HSC3 cells were transfected with siRNA and were then incubated for 48 h. Cells were pretreated with 25 µg/mL of nystatin for 30 min before treatment with 100 µg/mL of CTX for the indicated times. Total EGFR protein expression was analysed via Western blotting. CHX was added 1 h before nystatin treatment. (F) Effects of nystatin on CTX-induced apoptosis. HSC3 cells were transfected with siRNA and then incubated for 48 h. Cells were pretreated with 25 µg/mL of nystatin for 30 min before treatment with 100 µg/mL of CTX in a serum-free medium for 12 h. Apoptosis was analysed using Annexin V-APC and 7-AAD. * p < 0.001; n.s., not significant.