Journal: bioRxiv

Article Title: Evolution of hominin detoxification: Neanderthal and modern human AHR respond similarly to TCDD

doi: 10.1101/445239

Figure Lengend Snippet: Dose-response relation observed for TCDD-induced CYP1A1 mRNA expression in HeLa human cervix epithelial adenocarcinoma cells transfected with an expression construct carrying a cDNA copy of the modern human or Neanderthal AHR mRNA, respectively. CYP1A1 and β-actin (ACTB) mRNA expression was measured using qPCR after 40-44 hours of exposure to TCDD. CYP1A1 expression was normalized for ACTB expression. The CYP1A1/ACTB ratios where then normalized to the basal level for the modern human AHR and all other values expressed as a fold value relative to this value set at 1, and plotted against the TCDD concentration in the culture medium during exposure. Effective concentration 50% (EC 50 ), basal and maximal values for TCDD-induced CYP1A1 mRNA expression were calculated by fitting the one-site receptor-ligand binding equation (see Methods) to triplicate experimental data, which showed that only for the extrapolated maximal CYP1A1 expression level there was a statistical significant (p < 0.05) difference between the cells transfected with the modern human and the Neanderthal AHR, not for the basal level and EC 50 .

Article Snippet: Both 5’-UTR variants were generated by PCR using Phusion High-Fidelity DNA polymerase (New England Biolabs M0530) and as a template the full-length AHR cDNA clone SC119159 purchased from Origene (delivered in vector pCMV6-XL4; distributor Acris Antibodies GmbH, Herford, Germany).

Techniques: Expressing, Transfection, Construct, Concentration Assay, Ligand Binding Assay

Journal: Nature Communications

Article Title: Sustained activation of the Aryl hydrocarbon Receptor transcription factor promotes resistance to BRAF-inhibitors in melanoma

doi: 10.1038/s41467-018-06951-2

Figure Lengend Snippet: Long-term canonical activation of AhR drives melanoma resistance to BRAFi. a Graphical representation of AhR function controlling melanoma cell sensitivity or resistance during BRAFi treatment. A high level of heterogeneity is observed among melanomas with a high proportion of highly differentiated and β-cells sensitive to BRAFi (induction of pigmentation by AhR: β signature) and a weak number of undifferentiated and α-cells resistant to BRAFi (induction of α signature and resistance genes). These persister cells constitute a cell reservoir leading to melanoma relapse. b Graphical model of AhR activation by BRAFi and α-ligands, with α-ligands dictating melanoma resistance. c Expression heatmap for resistant genes in 501Mel cells treated for 7–14 days with TCDD (10 nM). d 501 melanoma cells (501Mel) were pre-treated daily or not for 2 weeks with TCDD (10 nM) and treated 4 days with increasing concentrations of Vem in order to establish cell density measurements and calculate IC50 (sensitivity to Vem). Values, calculated with GraphPad (PRISM6.0 ® ), represent the IC50 of Vem for control cells (without TCDD pre-treatment) or after 2 weeks of TCDD. e Expression heatmap for β-, α-, and resistance genes in 501Mel cells invalidated or not for AhR by CRISPR/Cas9 before or 48 h after treatment with Vem (1 μM). f Expression Heatmap for β-, α-, and resistance genes in 501Mel and SKMEL28 (R) cells knocked-down for AhR or ARNT using siRNA. The human silhouettes have been adapted (change of color background) from Servier Medical Art, licensed under a CC BY 3.0 FR [ https://smart.servier.com/smart_image/shape-29/ ]

Article Snippet: Plasmids encoding shRNA targeting human AhR (TL320259, 29mer shRNA constructs in lentiviral GFP vector) were purchased from Origene, Rockville, MD.

Techniques: Activation Assay, Expressing, Control, CRISPR