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. 2018 May 7;19(5):1388. doi: 10.3390/ijms19051388

Figure 1.

Figure 1

AHR downregulation reduces irregular colony formation in 3D Matrigel cultures and inhibits invasion in Boyden chambers. (A) Wild-type BP1 and Hs578T cells were co-transfected with AHRE-driven firefly luciferase reporter (pGudLuc), phRL-TK (renilla), and pcDNA control vector, AHRR plasmid, control scrambled siRNA, or AHR-specific siRNA as indicated and incubated for 24–48 h. Hs578T cells transduced with a CRISPR-Cas9 control vector or with an AHR-specific CRISPR-Cas9 construct were similarly transfected with pGudLuc and phRL-TK. Cells were harvested and pGudLuc luciferase activity normalized to renilla luminescence. Data from 3–5 independent experiments are expressed as normalized mean luciferase activity + SE. * p < 0.02, ** p < 0.01, *** p < 0.001 relative to controls using the Student’s t-test. (B) BP1 and Hs578T cells were transfected with pcDNA control plasmid, AHRR, control scrambled siRNA, or AHR-specific siRNA plasmids and plated 24 h later in 3D Matrigel cultures in duplicate wells. Hs578T cells transduced with a CRISPR-Cas9 control vector or with an AHR-specific CRISPR-Cas9 construct were similarly cultured in Matrigel. Images were taken on days 4–7 of culture at 400× magnification and are representative of a minimum of two independent experiments with duplicates in each experiment. (C) BP1 (top) and Hs578T (bottom) cells were transfected with scrambled control siRNA or AHR-specific siRNA for 24 h before serum starvation for 18 h. Cells were harvested, counted, resuspended in serum-free media, and plated in triplicate in the upper chamber of Boyden chambers. Serum-containing, complete medium was placed in the lower chamber. Chambers were separated by 8 μM Matrigel-coated membranes. Invasive cells in the lower chamber of individual wells were dissociated from the membrane 48 h later, lysed and stained with CyQuant GR dye and fluorescence quantified. Data pooled from 4–5 independent experiments are presented as the mean percent invasion normalized to untransfected controls + SE, * p < 0.05 using the Student’s t-test as compared with the scrambled siRNA-transfected control group.