Supplementary Figure 1: PrimPol mRNA and protein levels in the cell cycle. Chromatin association in response to DNA damage.
From: Repriming of DNA synthesis at stalled replication forks by human PrimPol
(a) Left panel: quantification of PrimPol mRNA levels by RT-PCR in cells synchronized at the indicated cell cycle stages. A, asynchronous cell culture. Right panel: PrimPol total protein levels, detected by immunoblot in the same cell populations. Mek2 serves as loading control. DNA content profile for each population is shown. (b) Histograms showing the ratio of chromatin-associated vs. soluble PrimPol protein in HeLa and U2OS cells exposed to the indicated treatment (Materials and Methods) and quantified from immunoblot signals such as those shown in main Figure 1b. In each case, chromatin/soluble PrimPol ratios are expressed as fold-change relative to the ratio obtained in untreated cells. (c) Immunoblots showing the distribution of PrimPol in soluble (S) vs. chromatin-bound (C) fractions in HeLa cells in the absence (lanes 1–6) or presence (lanes 7–12) of 2 mM HU. When indicated, cells were treated with 7.5 mM caffeine or 0.4 mM UCN01. Mek2 and CTCF are shown as controls of the soluble and chromatin fractions, respectively. (d) Recruitment of GFP-PrimPol to sites of laser-induced DNA damage in U2OS cell nuclei (depicted as dashed yellow lines in the leftmost images). Time lapsed between laser irradiation and image acquisition is indicated in minutes. GFP-53BP1 and GFP-H2AB were used as positive and negative control, respectively. Scale bar, 10 μm. Data are pooled from 3 experiments (left panel) and representative of 3 experiments (right panel) (a), pooled from 3 experiments for each cell line (b), representative of 3 experiments (c), representative of 16 laser-responsive cells from a total of 54 irradiated cells (PrimPol), 16 laser-responsive cells from a total of 35 irradiated cells (53BP1), 12 irradiated cells (H2AB) (d). Error bars, s.d. (a,b).
