Fig. 1: Hypoxia triggers PCNA monoubiquitination and increases expression of TLS DNA polymerases.

A HEK293FT cells were subjected to hypoxia (0.5% Oxygen) for 4 or 12 h. Chromatin-bound and soluble proteins were extracted, and analyzed by immunoblotting after running it on 4–20% ExpressPlus™ PAGE gel (Genscript) in SDS-MOPS buffer. B As in A, except that the cells were also subjected to hypoxia for 12 h, followed by 1 h of reoxygenation. C MCF7 and MRC5sv cells were subjected to 24 h of hypoxia (0.5% oxygen), after which total cell proteins were fractionated and immunoblotted for PCNA and mUb-PCNA. D HEK293FT cells were treated with siRNA against human RAD18 (50 nM) or its non-targeting control for 48 h and then subjected to hypoxia (0.5% oxygen) for 24 h after which its chromatin-bound and soluble proteins were analyzed by immunblotting for the presence of the indicated proteins, as described for panels A and B. E Expression data obtained from a free access data source, analyzed using Genevestigator software. Effect size threshold was 1.5-fold change (0.6 in log2 scale), and FDR threshold was 0.05 (all had an FDR score under 0.01, and almost all under 0.001). A Human Pulmonary microvascular endothelium (HPME) cell line was subjected to hypoxia (1% oxygen for 48 h; two samples) compared to untreated samples (three samples in normoxia). The effect of hypoxia on POLI, REV3L and PRIMPOL is highlighted. F HEK293FT and MCF7 cells were subjected to hypoxia with and without reoxygenation of 1 h, and analyzed by Western blot for the presence of DNA polymerase η.