APOBEC3A drives deaminase mutagenesis in human gastric epithelium

Overview of conditional APOBEC (APOBEC3A or APOBEC3B) overexpression models with human gastric organoids. (A) Experimental design of the study. Schematic illustration of genetic engineering, cloning, and sequencing (whole-genome sequencing, duplex sequencing, and bulk RNA-seq) is shown. Dox, doxycycline. (B) Changes of morphology and mCherry fluorescence during 48 h with 0.1 µg/mL doxycycline treatment; (top) hGO_iA3A lines, and (bottom) hGO_iA3B lines. Scale bars represent 1 mm. (C) Inducible expression of APOBEC3A (A3A) or APOBEC3B (A3B) enzymes in hGO_iA3A and hGO_iA3B lines, respectively, following 3 µg/mL doxycycline treatment for 48 h. (D) Expression levels of APOBEC3A (A3A) or APOBEC3B (A3B) in each line following 0.1 µg/mL and 3 µg/mL doxycycline treatment for 48 h; (left) hGO_iA3A lines (n = 3 per condition), and (right) hGO_iA3B lines (n = 3 per condition). Data are presented as mean ± 95% confidence interval. Statistical significance was determined using a t-test: (*) P < 0.05, (**) P < 0.005, (****) P < 0.00005. (E) Changes of viability of each line following 0.1 µg/mL and 3 µg/mL doxycycline treatment for 48 h (n = 4 per each condition). Data are presented as mean ± 95% confidence interval. (F) Differentially expressed genes upon APOBEC (A3A or A3B) overexpression following 0.1 µg/mL doxycycline treatment for 48 h; (top) hGO_iA3A line, and (bottom) hGO_iA3B line. adj.pval, adjusted P-value. (G) Subcellular localization of overexpressed A3A and A3B following 3 µg/mL doxycycline treatment for 48 h in each line; (left) hGO_iA3A line, and (right) hGO_iA3B line. Scale bars represent 50 µm. (H) γ-H2AX foci following 3 µg/mL doxycycline treatment for 48 h in each line; (left) hGO_iA3A line, and (right) hGO_iA3B line. Scale bars represent 50 µm.