Figure 10

Lack of AhR increases caveolae-dependent endocytosis. (A) Basal T-FGM AhR+/+ and AhR?/? fibroblasts and T-FGM AhR+/+ cells transfected with a si-AhR were grown on glass coverslips for 24 h. Cells were then incubated with serum-free medium for 2 h and with BSA-FITC for 1, 2 or 4 h. Cultures were observed in a Fluoview F1000 fluorescent confocal microscope. Cell nuclei were stained with Hoechst 33342. (B) Total fluorescence per cell was digitally calculated with the ImageJ software and represented for each treatment. (C) The same assay was performed in dermal fibroblasts from AhR+/+ and AhR?/? newborn mice measuring endocytosis after 4 h of treatment. (D) Total fluorescence per cell was digitally calculated with the ImageJ software. (E) T-FGM cells of both genotypes were treated with 10 mM M?CD, 15 mg/ml nystatin or solvent (control) for 1 h prior to the incubation with BSA-FITC for 4 h. Endocytosis was quantified and analyzed as indicated above. (F) T-FGM AhR+/+ and AhR?/? cells were pre-treated with 100 mM cholesterol plus 2.5 mM M?CD (choles) or solvent (control) for 16 h prior to the incubation with BSA-FITC for 4 h. Endocytosis was quantified and analyzed as indicated above. Data are shown as mean ± s.d. from experiments performed in duplicate in three independent cultures.