Supplementary Figure 3: Defective fork progression, inefficient fork restart and dormant origin activation upon PrimPol downregulation.

(a) Immunoblots showing the downregulation of endogenous PrimPol in HeLa-shPrimPol cells upon addition of Dox (lanes 1–2) and the efficient expression of the exogenous versions of the enzyme, detected with anti-PrimPol and anti-V5 (lanes 3–6). The asterisk marks a species cross-reacting with the anti-V5 antibody. Mek2 is shown as loading control. (b) Schematic of the labeling assay in which the CldU and IdU pulses are separated by a 5h HU treatment. Bottom, flow cytometry profiles of BrdU incorporation in asynchronous (Asy) or HU-treated cells. (c) Left panel: Histograms showing the percentage of fork restart after HU treatment in HeLa-shPrimPol cells with or without Dox. When indicated, plasmids expressing V5-tagged PrimPol (either WT or AxA mutant) were transfected. Right panel: Percentage of fork restart after HU in MEFs derived from WT or PrimPol KO mice. (d) Immunoblots comparing PrimPol levels in HDF-shPrimPol cells incubated with or without Dox for 72 h (lanes 4 and 5). Lanes 1–3 show serial dilutions (12.5, 25, 50%) of the sample analyzed in lane 4, for quantification purposes. (e,f) Scatter plots showing FR (e) or IOD (f) values in HDF-shPrimPol with or without Dox. (g) Percentage of fork rescue after UV irradiation (30 J/m2) in HDF-shPrimPol cells with or without Dox. Data are representative of 3 experiments (a,b), pooled from 2 (HeLa-shPrimPol) or 3 experiments (MEFs) (c), representative of 2 experiments (d), pooled from 2 experiments (e–g). Error bars, s.d. **P<0.01, ***P<0.001 (Student's t-test), NS, not significative (c,g). Horizontal lines represent median, ***P<0.001 (Mann-Whitney test) (e,f).