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. 2020 May 21;10:8477. doi: 10.1038/s41598-020-65414-1

Figure 1.

Figure 1

MMP1 levels, but not MMP2 or MMP9 levels are regulated by the AHR in Graves’ orbital fibroblasts (GOFs). GOFs were treated with non-specific siRNA (control) or AHR siRNA for 48 hours. Afterwards, cells were incubated with 0.1% FBS DMEM medium containing either vehicle (DMSO), TGFβ, TGFβ + 0.1 μM FICZ or TGFβ + 1 μM FICZ for 24 hours. Cell culture supernatants were then collected and analyzed by fluorescent based immunoassay (Luminex) for MMP1 (a), MMP2 (b) or MMP9 (c). MMP1 levels were reduced by TGFβ and increased by FICZ. AHR siRNA attenuated MMP1 induction by FICZ. MMP2 and MMP9 levels were not significantly altered by any treatments or by AHR siRNA. To confirm that FICZ activated AHR dependent gene expression and AHR siRNA successfully blocked AHR dependent gene expression, canonical AHR dependent genes were analyzed by qPCR in samples treated with vehicle, TGFβ or TGFβ + 1 μM FICZ for 24 hours. Total RNA was isolated and analyzed by RT-qPCR. Normalized levels of CYP1A1 mRNA (d), CYP1B1 mRNA (e) and AHRR mRNA (f) are shown. FICZ significantly induced expression of all three canonical AHR dependent genes in GOFs while AHR siRNA dramatically attenuated the effect of FICZ on gene expression. The experiment was performed in 3 different strains of GOFs. ##p < 0.01, ###p < 0.001 versus vehicle treatment. **p < 0.01 in AHR vs control siRNA samples with the same treatment.