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. Author manuscript; available in PMC: 2021 Apr 21.
Published in final edited form as: J Occup Environ Med. 2019 Dec;61(Suppl 12):S82–S89. doi: 10.1097/JOM.0000000000001691

Figure 2: AHR siRNA reduces AHR protein levels and HpCDD-mediated induction of CYP1B1 mRNA.

Figure 2:

(A) Control or pCMV-AHR plasmid DNA were introduced into HEK293FT cells (left panels) and human lung fibroblasts (right panels) were treated with control or AHR specific siRNA (2 different target sequences) for 72 hours. Then, protein was prepared and analyzed by Western blot for AHR and total actin (loading control). 293FT cells show robust expression of AHR with pCMV-AHR. In HLFs, AHR siRNA #1 reduced AHR protein levels to less than 10% of control siRNA levels and AHR siRNA #2 reduced AHR protein levels to less than 5% of control. (B) HLFs treated with the siRNAs as described in (A) were then exposed to vehicle (DMSO) or BghiP or HpCDD at the doses listed. After 24 hours, total RNA was collected and analyzed by RT-qPCR for the AHR-dependent mRNA, CYP1B1 and reference mRNAs, HPRT1 and TBP. With control siRNA treated cells, CYP1B1 levels were not significantly altered by either 0.2 or 1 µM BghiP, however, both 200 pM and 1 nM HpCDD led to a significant increase in CYP1B1 levels. In samples treated with either AHR siRNA, CYP1B1 induction was dramatically attenuated. The experiment was performed in 2 different HLF strains in triplicate with similar results in both strains. Representative data from 1 strain presented as means ± standard error, ** = p < 0.01, Two-way-ANOVA.