Extended Data Fig. 4: Knockout of Cdc7.

a-c, CRISPR/Cas9-mediated knockout of Cdc7 in primary mouse fibroblasts (MEFs). Cdc7^AID/AID/Tir1 MEFs were transduced with viruses encoding control sgRNA (sgCTR) or anti-Cdc7 sgRNA (sgCdc7). a, Whole cell lysates were immunoblotted and probed with an anti-Flag antibody to detect Flag-tagged endogenous Cdc7. Day 0, cells at the end of selection; Day 5, cells after 5 days of culture. b, Cell cycle analysis of cells from a. Cells were pulsed with BrdU, stained with an anti-BrdU antibody and propidium iodide and analyzed by flow cytometry. c, Doubling times. d–f, CRISPR/Cas9-mediated knockout of Cdc7 in MEFs immortalized by dominant-negative p53. d, Immunoblotting as in a. Polyclonal, total polyclonal cell population after transduction with sgRNAs; selected, further purified population containing over 90% of Cdc7 frame-shift alleles. e, cell cycle analysis (polyclonal population), f, doubling times (polyclonal population). g, h CRISPR/Cas9-mediated knockout of Cdc7 in keratinocytes; g, immunoblotting, h, doubling time. i–j, CRISPR/Cas9-mediated knockout of CDC7 in human BJ foreskin fibroblasts. i, Immunoblotting with anti-CDC7 antibody, j, Cell cycle analysis. k–p, Acute ablation of Cdc7 in ESC. Cdc7^Flox/Flox (Cdc7^F/F) and Cdc7^+/+ ESC were engineered to stably express dominant-negative p53 (to prevent p53-dependent cell death upon shutdown of Cdc7³³). Cells were transduced with lentiviruses encoding Cre-recombinase, or mock transduced. k, Cells were harvested at days 2 and 3, and extracts immunoblotted with an anti-Cdc7 antibody. l, m, at day 3, cells were stained with propidium iodide (l), or pulsed with BrdU and stained with propidium iodide and an anti-BrdU antibody (m) and analyzed by flow cytometry. n, Cells were cultured in the presence of BrdU for 15 h, stained with an anti-BrdU antibody and the percentage of BrdU⁺ cells was evaluated by flow cytometry. Note that 98% of Cdc7-deleted cells (and the same fraction of Cdc7^+/+ cells) incorporated BrdU, indicating active DNA synthesis. FSC, forward scatter. o, Cells were stained with Annexin V and propidium iodide (PI) and analyzed by flow cytometry. Shown are percentages of viable (Annexin V^–/PI^–), early apoptotic (Annexin V⁺/PI^–), late apoptotic (Annexin V⁺/PI⁺) and necrotic (Annexin V^–/PI⁺) cells. p, Cdk1 was immunoprecipitated (IP-Cdk1) from cells and used for kinase reactions with [γ³²P]-ATP and histone H1 as a substrate. IgG, control immunoprecipitation with IgG. Note that ablation of Cdc7 resulted in modestly elevated levels of Cdk1 kinase - like an acute degradation of Cdc7 in Cdc7^AID/AID/Tir1 ESC (Extended Data Fig. 7b). Gapdh was used as loading control. c, f, o, show means; p-values by two-sided t-test; error bars, SD; n = 3 independent replicates; other panels show representative results (out of 2).