HCT116 cells surviving PARP1 depletion activate innate immune signaling.

<p>(A-B) RNA-Seq data from HCT116^EV and HCT116^{<i>PARP1</i>-/-} cells (clones C2 and C4) was analyzed using Gene Set Enrichment Analysis (GSEA). The top category of differentially expressed genes was “Interferon Alpha Response”. The enrichment plot is shown in (A) and the heat map for the 97 mRNAs in this category in shown in (B). Map was generated using Cufflinks software (version 2.2.1) and shows absolute expression values independently normalized and analyzed for each comparison pair (EV/C2 and EV/C4). (C) The same RNA-Seq dataset was analyzed using Ingenuity Pathway Analysis (IPA). A representative plot highlighting enrichment for Interferon-Stimulated Genes (ISGs) is shown. (D) The induction of multiple ISGs observed by RNA-Seq was confirmed by q-RT-PCR. Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–4 independent experiments. (E-F) The induction of factors involved in the sensing/signaling of cytoplasmic nucleic acids observed by RNA-Seq was confirmed by Q-RT-PCR (E). Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–3 independent experiments. Protein expression for the same factors was assessed by immunoblotting (F). Blots are representative of 2–3 independent experiments. (G) Fixed cells were stained with an antibody to IRF3 and counterstained with DAPI. Images are representative of 5 random fields per slide. The experiment was repeated twice with similar results.