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. 2022 Nov 7;10(11):e004974. doi: 10.1136/jitc-2022-004974

Figure 5.

Figure 5

RNA editing of immunogenic RNA increases with DNMTi in human OC cells. (A–C) TykNu and isogenic CRISPR-edited Hey cell lines (TP53 R175H mutant/TP53 wild-type) were treated for five consecutive days with Mock (PBS) or 5-Aza (500 nM) and then harvested on day 9. Another set of flasks were stimulated with IFN-β for 24 hours prior to harvesting. Protein was isolated and immunoblotted for ADAR1 and β-actin (loading control). (D–F) TykNu and isogenic CRISPR-edited Hey cell lines (TP53 R175H mutant/TP53 wild-type) were treated for five consecutive days with Mock (PBS) or 5-Aza (500 nM) and then harvested on day 9. RNA was isolated from three treatment replicates each, and Illumina sequencing libraries were prepared and sequenced, resulting in paired-end, stranded reads. (D) Alu editing index and LTR editing for TykNu TP53 R175H mutant. (E) Alu editing index and LTR editing for Hey TP53 R175H mutant. (F) Alu editing index and LTR editing for Hey TP53 wild-type. (G) Colony formation assays showing Mock, IFN-β Dac±IFN-β, 5-Aza±IFN-β, and across TykNu shSCR or shADAR1 (each condition performed in triplicate). (H) Colony formation assays showing Mock, IFN-β Dac±IFN-β, 5-Aza±IFN-β, and across Hey TP53 mutant shSCR or shADAR1 (each condition performed in triplicate). Unpaired t-tests were performed for statistical significance for figure 1D, F. *P<0.05; **p<0.01. ADAR1, adenosine deaminase 1; 5-AZA, 5-zaacytidine; Dac, 2’-deoxy-5-azacytidine; DNMTi, DNA methyltransferase inhibitor; IFN, interferon; LTR, long-terminal repeat; OC, ovarian cancer; PBS, phosphate-buffered saline.