Fig. 4: TLS is involved in global DNA replication during hypoxia.

A MCF7 cells were transfected with the indicated siRNA (50 nM) and incubated for 48 h. After that, the cells were exposed to hypoxia (0.5% oxygen) for 16 h, treated with BrdU for two hours, and fixated with ethanol. BrdU incorporation was analyzed using Roche’s ELISA, BrdU (colorimetric) kit according to manufacturer’s protocol. B MEFs with either a Pcna-K164R mutation or WT PCNA, were grown under hypoxia, and BrdU incorporation was measured as described in A. C XP12RO human cells were treated with the indicated siRNAs, followed by hypoxia and then viability analysis using the CellTiter-Glo® Luminescence Assay. D,E HEK293FT cells were subjected to 16 h hypoxia (0.5% oxygen) D or transfected with HA-HIF1A^P402A/P564A under normoxia E, and the iPOND protocol was then conducted. In short, the cells were supplemented with EdU or DMSO, and after 60 min were crosslinked, permeabilized, and biotin was added for a click reaction with the EdU. The cells were lysed, and Streptavidin beads were added to capture the biotin. After a wash, the beads were boiled in an elution buffer, and the samples were analyzed by mass spectrometry. Hypoxia-specific enrichments of proteins on nascent DNA D was calculated from the MS results (Data Set 1) as: (Hypoxia Pulse/Chase LFQ-intensity ratio)/(Normoxia Pulse/Chase LFQ-intensity ratio). HIF1α-specific enrichments of proteins on nascent DNA E was calculated from the MS results (Data Set 2) as: (HIF1α Pulse/Chase LFQ-intensity ratio)/(Empty Vector Pulse/Chase LFQ-intensity ratio). The two datasets were deposited in the Figshare Repository at https://doi.org/10.6084/m9.figshare.27061339. The lists were filtered for proteins identified based on ≥10 unique peptides with ≥10% unique sequence coverage, and ranked in descending protein enrichment ratio. The 10 highest ranking proteins enriched on nascent DNA under hypoxia D or in normoxic cells expressing stable HIF1α E are presented.