Fig. 2

ADAR1 expression is required for DEspR protein expression in Panc1 and MiaPaCA2 tumor cells (TCs). a Flow cytometry analysis of permeabilized Panc1-TCs showing baseline 98% ADAR1+/DEspR+ co-expression. CRISPR/Cas9-knockout (KO) TCs at passage (p)-3 exhibited 0.22% ADAR1+, 98% DEspR+ expression; at p4: 0.01% DEspR+/ADAR1+ (100,000 cells/duplicate); see Table S1. b Quantitative western-blot analyses of p5 ADAR1-KO Panc1-TCs showing decreased ADAR1(A) (18.5% ± 4.1%), DEspR(D) (9.4% ± 1.3%), and Mcl-1(M) (10.4% ± 1.8%) compared to wild type (WT) Panc1-TCs (100%-reference). [Western blots in Fig. S3]. c Flow cytometry study of permeabilized MiaPaCa2-TCs showing 97.5% baseline co-expression of ADAR1+/DEspR+. CRISPR/Cas9-ADAR1-KO TCs had 4.73% ADAR1 + expression (94.6% DEspR+) by p3; 0.02% DEspR+ADAR1+ at p4 (100,000 cells/n = 2); Table S1. d Quantitative western-blot analyses of ADAR1-KO MiaPaCa2 TCs showing decreased ADAR1(A) (9.0% ± 1.3%), DEspR(D) (17.5% ± 6.4%), and Mcl-1(M) (32.9% ± 5.6%) compared to WT MiaPaCa2-TCs (100%-reference). [Western blots in Fig. S3] e Mock-control for ADAR-1 KO using murine-specific CRISPR/Cas9-ADAR1-KO construct showing no knockout of human-ADAR1 and no loss of DEspR expression by p4 despite successful transfection (reporter-GFP+ expression). f Representative phase-contrast images at 5-days post-seeding showing decreased survival/growth and tumorsphere-formation in low adherence conditions of p5 ADAR1-KO vs WT Panc1 and MiaPaCa2 TCs. Graph of number(#) of tumorspheres > 50 μm-diameter on day-1, − 3, and − 5 post-seeding comparing ADAR1-WT and ADAR1-KO tumor cells (***p = 0.000036) and MiaPaCa2-TCs (****p = 0.000002); paired two-tailed t-test, Bonferroni-Dunn correction, 5-replicates/point