Figure 4. Reverse transcriptase inhibition leads to decreased cytoplasmic DNA, and increased RNA:DNA hybrid intermediates.

A-C, Gene set enrichment analysis (GSEA) comparing 3TC patient pre-treatment and on treatment biopsy RNA-seq (A) L1, (B) Satellite, and (C) ERV repeats. Repeats were ranked by Log2(Fold Change, on-treatment vs pre-treatment tumor sample) for GSEA. Significant decrease in L1, Satellite, and ERV repeat levels after treatment with 3TC shown in patient biopsies with p value and corrected q value shown (See Supplementary Methods). D, Volcano plot representing differential expression (Log2FC) vs Significance (-LogP) of coding genes in tumor biopsies before treatment, and after treatment with 3TC. Interferon response genes (blue) are significantly upregulated after treatment with 3TC. E, Immunofluorescence (IF) of cytoplasmic dsDNA in P53-Mut tumorspheres treated with DMSO, or 3TC (1 µM) for 7 days. Quantification of cytoplasmic IF signal indicates significant decrease in cytoplasmic dsDNA in response to 3TC. Significance determined by two tailed t-test with Welch’s correction: **** p < 0.0001. F, IF of cytoplasmic RNA:DNA hybrids in P53-Mut tumorspheres treated with DMSO or 3TC (10 µM) for 7 days. Quantification of cytoplasmic IF signal indicates significant increase in cytoplasmic RNA:DNA in response to 3TC. Significance determined by two tailed t-test with Welch’s correction: **** p < 0.0001. G-I, Rescue of effects of 3TC on cell migration by STING knockdown. (G) Western blot analysis confirms knockdown of STING (TMEM173) protein using pooled siRNA compared to non-targeting control in DLD1 cells. (H) Representative images of DLD1 cell migration with siRNA against STING (right) or Mock (left) and treated with DMSO or 3TC. (I) Quantification of DLD1 migrated cells in siRNA STING and 3TC experiments in (H). Statistical significance determined by student’s two tailed t-test as compared to non-targeting control siRNA treated with DMSO: ** p < 0.01.