Supplementary Figure 2: Slow cell proliferation and signs of chromosomal instability after PrimPol downregulation.
From: Repriming of DNA synthesis at stalled replication forks by human PrimPol
(a) Cell proliferation profiles of U2OS-shPrimPol with or without Dox. The efficiency of PrimPol downregulation at each time point is shown in immunoblots, with Mek2 as loading control. (b) Distribution of HeLa-shPrimPol cells in the different phases of the cell cycle, after 72 h in the absence or presence of Dox. Percentages of cells in each phase were calculated from DNA content profiles generated by flow cytometry after propidium iodide staining. (c) Flow cytometry detection of BrdU incorporation after a 30 min pulse with 10 μM BrdU in HeLa-shPrimPol with or without Dox. The histogram represents percentage of BrdU-positive cells. (d) Left: representative images of aberrant mitotic figures in HeLa-shPrimPol cells. Arrows mark the position of misaligned chromosomes in metaphase and lagging chromosomes in anaphase. Microtubules were stained with anti-α-tubulin (green) and anti-γ-tubulin (red). DNA was stained with DAPI (gray). Scale bar, 5 μm. Bar graphs indicate the percentage of abnormal metaphases or anaphases in the absence or presence of Dox. (e) Similar analysis as in (d), carried out in WT or PrimPol KO MEFs. (f) Representative images of chromosomal breaks and gaps in metaphase spreads of T lymphocytes derived from PrimPol KO mice. (g) Left, representative images of triradial and tetraradial chromosomal fusions in metaphase spreads of T lymphocytes derived from PrimPol KO mice. Histogram (right) shows the percentage of metaphases showing chromosome fusions in each case. Immunoblots are representative of 3 experiments that were pooled for growth curves (a), data are pooled from 5 experiments (b), flow cytometry plots are representative of 5 experiments, which are pooled in the histograms (c), images are representative of 4 experiments, combined in the histograms (d), data are pooled from 3 experiments (e), images are representative from the analysis of 80 metaphases per condition (2 mice per genotype) (f,g). Error bars, s.d. (b–e, g), *P<0.05, ***P<0.001. (Student's t-test) (d,e).
