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. 2023 Jan 27;13:1537. doi: 10.1038/s41598-023-28840-5

Figure 5.

Figure 5

Loss of DNA Methylation in fallopian tube cells leads to LINE-1 ORF1p expression and detection in conditioned media. FT cell lines were treated with DNMT inhibitors Decitabine or SGI-110 (5 µM) for 3, 5 or 7 days. DMSO was used as negative control. (A) DNMT1A protein expression (WB) and (B) LINE-1 methylation levels after Decitabine treatment. 5-methylcytosine levels of LINE-1 in genomic DNA were quantified and the optical density (OD) was measured at 450 nm. A significant decrease in DNMT1A levels and LINE-1 methylation (N = 3. ***p < 0.0002) was observed. (C) ORF1p protein expression (WB) in FT cells after Decitabine treatment. None of the lines expressed ORF1p prior to treatment but ORF1p was abundantly expressed in all the lines after 5 days of treatment. β-actin serves as the loading control. (D) Comparison of LINE-1 ORF1p expression in FT cells after Decitabine or SGI-110 treatment by WB. Both compounds are equally capable of inducing ORF1p expression as early as 3 days. (E) FT cells treated with Decitabine or SGI-110 were examined by immunofluorescence for the presence of ORF1p (10X objective). (F) ORF1p protein expression (WB) in conditioned media of FT cells after treatment with demethylating agents. COV318 was used as a positive control for ORF1p release.