Fig. 4

Aldh1a1 downmodulation reduces cancer stem-like and pluripotency markers in AhR knockdown melanoma cells. a sh-AhR and sh-AhR + sh-Aldh1a1 cells were stained for CD133⁺ (CD133-PE), CD44⁺ (CD44-PerCP) and CD29⁺ (CD29-FITC) and analyzed by flow cytometry. b, c The same number of sh-AhR and sh-AhR + sh-Aldh1a1 cells was grown in ultra-low adherence culture dishes. The size (b) and number (c) of the melanospheres formed were analyzed using a Cell-R microscope and the ImageJ software. d The mRNA expression levels of the pluripotency genes Sox2, Klf4, c-Myc and Tcf4 were analyzed by RT-qPCR using total RNA isolated from sh-AhR and sh-AhR + sh-Aldh1a1 cells. e sh-AhR and sh-AhR + sh-Aldh1a1 cells were transiently transfected with a Sox2 expression vector and the mRNA levels of Aldh1a1 quantified by RT-qPCR. The empty vector (E.V.) was also transfected to normalize gene expression. f sh-AhR and sh-AhR + sh-Aldh1a1 cells were transfected with a Sox2 expression vector and their migration potential determined in wound healing assays. RT-qPCR data were normalized by Gapdh expression and represented as 2^-ΔΔCt. A.U. arbitrary units. Measurements were done in triplicate in two different cultures of each cell line. At least 6 fields were analyzed for each wound in panel (f). Data are shown as mean ± SD. A.U. stands for arbitrary units. n.s. = statistically non-significant