Figure 2

Dioxin receptor expression modulates Cav-1 distribution in DRMs and at the rear of migrating fibroblasts. (A) Extracts from T-FGM AhR+/+ cells transfected with a scramble (si-scr) or with a specific siRNA for AhR (si-AhR) and extacts from T-FGM AhR?/? cells were analyzed by sucrose density gradient centrifugation. The presence of Cav-1 in each fraction of the gradient was analyzed by immunoblotting. Gapdh was used as a cytoplasmic control protein. Detergent resistant membrane microdomains (DRM) correspond to the lower density (upper) fractions of the gradient. The quantification of the percentage of Cav-1 located in DRM fractions with respect to the total amount of Cav-1 in a representative experiment is shown. Data were normalized to the T-FGM AhR+/+ si-scr control condition. (B) RNA interference against AhR efficiently down-modulated protein expression in T-FGM AhR+/+ fibroblasts as compared with non-specific scramble sequences (si-scr). (C) Basal AhR+/+ and AhR?/? T-FGM fibroblasts, AhR+/+ fibroblasts transfected with a siRNA for AhR and AhR?/? cells transfected with a pcDNA-AhR expression construct were grown to confluence and analyzed for directional migration using wound-healing assays. The same experiment was performed using dermal fibroblasts isolated from AhR+/+ and AhR?/? newborn mice (right). Cav-1 was detected by immunofluorescence in a Fluoview F1000 confocal microscope. Cell nuclei were stained with DAPI. Arrowheads indicate rear distribution of Cav-1 in AhR?/? cells. The experiments were done at least three times in independent T-FGM and dermal fibroblasts cultures.