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. Author manuscript; available in PMC: 2017 Oct 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2016 Aug 14;309:15–23. doi: 10.1016/j.taap.2016.08.011

Figure 1.

Figure 1

A. Schematic representation of pTRIPZ vector for doxycycline-inducible transgene expression. In this vector, the expression of transgenic human aryl hydrocarbon receptor fused to green fluorescent protein (AHR-GFP) is under the control of human cytomegalovirus (CMV) constitutive promoter plus tetracycline response element (TRE) promoter, which can be activated by reverse tetracycline transactivator 3 (rtTA3) in the presence of doxycycline. pTRIPZ vector contains the 5’ and 3’ long terminal repeats (LTRs), packaging signal (ψ), internal ribosomal entry site (IRES) and puromycin selectable marker (Puro). B. Levels of the AhR gene expression in the SKW 6.4, SKW-AHR+, HepG2 cell lines and human primary B lymphocytes. Total RNA was extracted from untreated SKW 6.4, SKW-AHR+, HepG2, human primary B cells (1×106 cells/ml) and 500 ng of total RNA were analyzed by RT-PCR for AhR mRNA. Steady-state mRNA levels of the AhR were normalized to the endogenous 18S ribosomal RNA. AhR mRNA in the SKW 6.4 cells was not detected (ND). Representative of 3 independent experiments. Data are presented as the mean ± SD. C. Western blot analysis of the AhR protein expression. Whole cell lysates from the SKW 6.4 (60 μg), SKW-AHR+ (60 μg), HepG2 (3 μg) and human primary B cells (20 μg) were subjected to electrophoresis, blotted, and stained as described under Materials and Methods. Lane 1: SKW 6.4; lane 2: SKW-AHR+; lane 3: human primary B lymphocytes; lane 4: HepG2. Ratio was determined by dividing AhR protein density by the amount of cellular protein loaded per lane. One representative result from three independent experiments is shown. D. Inducibility and stability of the AhR mRNA in the absence and presence of doxycycline. SKW-AHR+ cells (1×106 cells/ml) were treated with vehicle (0.02 % DMSO) or TCDD (30 nM) in the presence of doxycycline (0.2 μg/ml), harvested at 7 h and 96 h of culture and analyzed for levels of AhR mRNA expression. AhR mRNA levels were normalized to the endogenous 18S ribosomal RNA. Data are presented as the mean ± SD from 3 independent experiments.