Figure EV4. (related to Fig 4). PRIMPOL downregulation or genetic ablation impairs cell proliferation and DNA synthesis in the presence of ICLs.

1. Top, experimental design of the CellTiter‐Glo viability assays in U2OS‐shPRIMPOL cells after treatment with 1 µg/ml MMC (2 h) or TMP‐UVA (2 µM TMP, 2 h followed by 5 s irradiation). When indicated, PRIMPOL expression was downregulated by a specific shRNA. Bottom, immunoblot shows the levels of PrimPol in control cells or after the expression of shPRIMPOL for 3 or 6 days. Ponceau S staining is shown as loading control.
2. Histograms represent the total number of viable cells (average and SD of three assays) at the end of the experiment, expressed as percentage of the number of untreated cells. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA and Bonferroni post‐test. P‐values are indicated.
3. Immunoblots show levels of endogenous PrimPol and exogenous V5‐PrimPol expressed in KO cells. Tubulin is shown as loading control.
4. Flow cytometry profiles of BrdU incorporation (y‐axis) vs. DNA content (PI; x‐axis) in WT (blue) and PRIMPOL KO (orange) cells, either collected without treatment (UNT) or at the indicated times after exposure to UVA (5 s irradiation). The DNA content profiles are shown on top of the boxes (cell count). Dashed horizontal lines inside panels are included for comparative purposes between WT and KO BrdU incorporation levels.
5. Same as in D, in cells collected without treatment (UNT) or at the indicated times after exposure to 1 µg/ml MMC (2 h). Histograms (right panel) represent the percentage of cells in S‐phase in each condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis in all cases was conducted with one‐way ANOVA and Bonferroni post‐test. P‐values are indicated.

Source data are available online for this figure.