Fig. 4.

Both non-enzymatic and catalytic functions of ADAR1-p150 may be important to prevent cell lethality in cancer cell lines. a GFP control, WT ADAR1-p150, E912A ADAR1-p150, or WT ADAR1-p110 proteins were overexpressed in HCC366 (right) or NCI-H1650 cells (left) prior to transduction with lentivirus that co-expressed Cas9 and sgRNAs targeting GFP or ADAR. Protein lysates were collected 6 days after GFP or ADAR KO and were probed with antibodies against ADAR1, phospho-PKR, total PKR, and β-Actin (loading control) in HCC366 (left) or NCI-H1650 cells (right). The fold change in the phospho-PKR to total PKR ratio (P-PKR/Total PKR) relative to the corresponding GFP sgRNA control is shown in each lane (n = 2). b Cell viability of GFP control or ADAR1-overexpressing HCC366 cells was assessed by crystal violet staining 11–13 days after GFP or ADAR KO (using ADAR sg2) with CRISPR-Cas9. A representative image of crystal violet staining (left) and quantitation of cell viability (right) from two independent biological replicates are shown. Cell viability values were normalized to the GFP sg2 control within each pair of isogenic cell lines. c Cell viability of GFP-overexpressing (control) or ADAR1-overexpressing NCI-H1650 cells was assessed by crystal violet staining 13–16 days after GFP or ADAR KO (using ADAR sg2) with CRISPR-Cas9. A representative image of crystal violet staining (left) and quantitation of cell viability (right) from two independent biological replicates are shown. Cell viability values were normalized to the GFP sg2 control within each pair of isogenic cell lines. d Model of the pathways that mediate cell lethality and interferon-induced interferon production after ADAR deletion in cancer cell lines. Error bars represent standard deviation in all graphs