FIGURE 5.

Abl is required for the clathrin-mediated endocytosis. (A) Huh7.5 cells were treated with either DMSO or the indicated concentration of imatinib or trifluoperazine for 2 h at 37°C. Cells were further treated with membrane fluidity detection dye for 20 min at 25°C and then wavelength was detected. Generalized polarization (GP) = (I405-I460)/(I405+I460), where I405 and I460 are the fluorescence intensity measured under wavelength 405 and 460, respectively. The asterisks indicate significant differences (^∗∗P < 0.01; ^∗∗∗P < 0.001) from the value for the DMSO control. (B) Huh7.5 cells were incubated with serum-free DMEM in the presence of either DMSO or the indicated concentrations of imatinib or chlorpromazine. The cells were incubated with 10 μg/ml Texas Red-conjugated transferrin in serum-free DMEM supplied with 1% BSA for 1 h at 4°C. The cells were fixed in 4% paraformaldehyde. Fluorescence intensity was determined by measuring the internalized transferrin using Image J. (C) (Upper panel) Huh7.5 cells were treated with either DMSO or imatinib for 4 h at 37°C and then infected with Jc1 using an MOI of 5. The cells were then fixed in 4% paraformaldehyde and stained with anti-clathrin heavy chain and TRITC antibodies. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue). (Lower panel) Fluorescence intensity of clathrin was quantified by using ImageJ software. (D) (Upper panel) Huh7.5 cells were transfected with either negative siRNA or Abl-specific siRNAs for 48 h and then infected with Jc1 using MOI of 5. At 30 min post-infection, cells were fixed in 4% paraformaldehyde and stained with anti-clathrin heavy chain and TRITC antibodies. Cells were counterstained with DAPI to label nuclei. (Lower panel) Fluorescence intensity of clathrin was quantified by using ImageJ software.