Fig. 3

Aldh1a1 knockdown reduces the migratory and invasive phenotypes produced by AhR depletion. a sh-AhR B16F10 melanoma cells were stably transduced with a sh-RNA for Aldh1a1 to generate the sh-AhR + sh-Aldh1a1 cell line. sh-AhR and sh-AhR + sh-Aldh1a1 cells were analyzed for Aldh1a1 protein expression by immunoblotting and for Aldh1a1 activity by flow cytometry using the Aldefluor reagent kit. To normalize Aldh1a1 activity, measurements were taken in presence and absence of the Aldh1-specific inhibitor diethylamino-benzaldehyde (DEAB). Values were then referred to those obtained for sh-AhR + sh-Aldh1a1 cells. b sh-AhR and sh-AhR + sh-Aldh1a1 cells were grown to confluence and then used for wound healing migration assays. c Both cell lines were cultured in matrigel transwells and analyzed for their invasive potential by confocal microscopy. Cell invasion at a depth of 40 μm is shown. d Clone formation in 2-D was determined by growing sh-AhR and sh-AhR + sh-Aldh1a1 cells at low cell density in plain tissue culture dishes. Clones were counted as unexpanded (compact clones with a dense cellularity), expanded (cells spreading out with reduced cell-cell interactions) or intermediate clones. e Clones were photographed, counted and their spreading analyzed using the ImageJ software. Three levels of spreading were established for compact (unexpanded), intermediate and fully expanded clones. Bars correspond to 100 μm (b) and 50 μm (d). A.U. arbitrary units. Experiments were done in duplicate or triplicate in three different cultures. At least 6 fields were analyzed for each wound in panel B. Data are shown as mean ± SD. A.U. stands for arbitrary units