Figure 9

AhR modulates basal cholesterol levels in fibroblasts. (A) T-FGM AhR+/+ and AhR?/? cultures were stained with the cholesterol dye M1 ganglioside (GM1) and membrane microdomains analyzed by fluorescence confocal microscopy. (B,C) T-FGM cells (B) and primary dermal fibroblasts (C) of both genotypes were grown on glass coverslips, fixed and stained with the cholesterol-binding antibiotic filipin III in order to detect endogenous free cholesterol. Stained cells were analyzed by flow cytometry and the fluorescence intensity profiles represented. (D,E) T-FGM AhR+/+ and AhR?/? cells were incubated for 16 h with 10 mM M?CD, 100 mM cholesterol plus 2.5 mM M?CD (cholesterol) or solvent and analyzed for cholesterol content by flow cytometry as indicated above. Fluorescence profiles were compared graphically. (F) T-FGM cells of both genotypes were grown to confluence and treated with M?CD or cholesterol as indicated above. Wound healing was used to induce directional migration. Cav-1 distribution was analyzed by fluorescence confocal microscopy in a Fluoview F1000 equipment. DAPI staining was used to label cell nuclei. Arrowheads mark Cav-1 location. The experiments were done in duplicate in two cultures of each genotype.