Fig. 1

AhR expression represses Aldh1a1 in mouse and human melanoma cells. a B16F10-derived cell lines were previously generated to stably express very low (sh-AhR), basal (B16F10) or a constitutively active (CA-AhR) AhR [23]. They were analyzed for Aldh1a1 mRNA expression by RT-qPCR and for protein expression by immunoblotting. b Aldh1a1 activity was measured by flow cytometry in sh-AhR, B16F10 and CA-AhR cells 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 wild type B16F10 cells. c, d B16F10 cells were treated with the AhR endogenous ligand FICZ (5 nM) and the mRNA levels of Aldh1a1 (c) and Cyp1a1 (d) determined by RT-qPCR. e A375 human melanoma cells were transfected with a sh-AHR to downmodulate their high basal receptor levels (upper). C8161 human melanoma cells were transfected with an AHR expression vector (AHR) to increase their low receptor levels. f, g ALDH1A1 mRNA and protein amounts were determined in A375 (f) and C8161 (g) cell lines by RT-qPCR and immunoblotting, respectively. The expression of β-Actin was used to normalize protein loading. RT-qPCR data were normalized by Gapdh expression and represented as 2^-ΔΔCt. The B16F10 cell line was also transduced with a retrovirus containing the empty vectors used to produce the sh-AhR and CA-AhR lines. Determinations were done in triplicate in at least two different cell cultures. Data are shown as mean ± SD