Fig 2: Hypoxia-induced replication stress leads to increased APOBEC expression but decreased deamination. (A) RKO cells were exposed to the indicated oxygen concentration or HU (2 mM, 20 h) for the indicated times and western blotting was carried out; ß-actin was used as a loading control. (B, C) RKO cells were exposed to the oxygen concentrations indicated or HU (2 mM) for 20 h. The mRNA levels of the indicated APOBECs, A3D (B) and A3H (C), as a relative fold change to normoxic (21% O2) levels are shown. (D) RKO cells were exposed to the oxygen concentrations indicated for the times shown or HU (2 mM, 20 h) followed by western blotting as indicated. (E, F) RKO cells were treated with siRNA to p53 (sip53), or a scramble control (siSCR) followed by exposure to <0.1% O2 for 20 h. mRNA levels of A3D (E) and A3H (F) are shown relative to the normoxic control. (G) RKO cells were exposed to the oxygen concentrations indicated for 20 h or HU (2 mM, 20 h). The mRNA level of A3B as a relative fold change to normoxic (21% O2) level is shown. (H) RKO cells were exposed to the conditions indicated or HU (2 mM, 20 h) followed by western blotting. For clarity, long and short exposures of A3B blot are shown. (I) RKO cells were treated with siRNA to p53 (sip53) or a scramble control (siSCR) (indicated as -) followed by exposure to the indicated oxygen concentration or HU (2 mM) for 20 h. Western blotting was carried out. (J) Spearman rank-order correlation between a signature of hypoxia-induced p53 response genes and APOBEC mRNA expression in Bladder Urothelial Carcinoma (BLCA), Breast Invasive Carcinoma (BRCA), Colorectal Adenocarcinoma (COAD), and Lung Adenocarcinoma (LUAD) from the TCGA harmonized dataset (see methods). For each gene the correlation values (top raw) and P values (bottom raw) are given. Color scale of correlation values is shown on the right of the heatmap. (K) RKO cells were exposed to the indicated oxygen concentrations or HU (2 mM) for 20 h followed by a deamination assay. The top band (S) is the substrate band and the bottom band (P) is the product band. (L) Quantification of data shown in part K where the average intensity of the product band (P) across three biological repeats is plotted relative to the intensity of the normoxic product band. Data from three separate experiments (n = 3) are displayed ± standard error of the mean (SEM) unless specified otherwise. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Cyclic hypoxia increases the expression and activity of A3B. (A) RKO cells were exposed to the oxygen concentrations indicated for the times shown or HU (2 mM, 20 h). Western blotting was carried out. (B) RKO cells were exposed to the indicated oxygen treatments for 20 h or HU (2 mM, 20 h) and co-stained for A3B and DAPI. The cells were visualized by immunofluorescence. (C) Quantification of A3B nuclear intensity from part B. (D) In vitro deamination assay measuring deamination activity in samples collected in the indicated hypoxic treatments for the indicated times or HU (2 mM, 20 h). The top band is the substrate band (S) and the bottom band is the product band (P). (E) Quantification of deamination in D. where the average intensity of the product band (P) (bottom) across 3 biological repeats is plotted relative to the intensity of the normoxic product band. (F) RKO cells were treated with an siRNA to A3B (siA3B) or a scramble (siSCR) (indicated by –) and then exposed to the hypoxic conditions indicated or HU (2 mM) for 20 h, followed by western blotting. (G) The samples from F. were also analyzed by deamination assay. The top band (S) is the substrate band and the bottom band (P) is the product band. (H) Quantification of data shown in part G where the average intensity of the product band (P) across 3 biological repeats is plotted relative to the intensity of the normoxic product band. (I) RKO cells were treated with siRNA to A3B (siA3B) or a a scramble control (siSCR) followed by exposure to cyclic hypoxia for 20 h. Cells were labeled with BrdU (20 µM) 1 h prior to collection and analyzed by FACS. Quantification of the data shows the percentage of cells in each phase of the cell cycle is shown. Exemplar FACs plots are shown in Figure S4D. (J) RKO cells were treated with siRNA to A3B (siA3B), or a scramble control (siSCR) followed by exposure to cyclic hypoxia or normoxia (21% O2) for 20 h. Western blotting was carried out. Data from three separate experiments (n = 3) are displayed ± standard error of the mean (SEM) unless specified otherwise. ** P < 0.01, *** P < 0.001.