Extended Data Fig. 6: Analyses of human CDK2-deficient cells.

a–d, Analyses of CDK2-knockout human mammary epithelial MCF10A cells. a, Whole cell lysates from parental (Control) and CDK2-knockout (sgCDK2) MCF10A cells were immunoblotted and probed with an anti-CDK2 antibody. b, CDK2^+/+ and CDK2-knockout (sgCDK2) MCF10A cells were cultured in the absence (Control) or presence of CDC7 inhibitor (XL-413) for 12 h. Cells were pulsed with BrdU, stained with an anti-BrdU antibody and propidium iodide and analyzed by flow cytometry. c, Parental and CDK2-knockout (sgCDK2) MCF10A cells were arrested in G0 by growth factor deprivation (Starved) and stimulated to re-enter the cell cycle by addition of growth factors (Released). Cells were pulsed with BrdU after 19 h, stained with propidium iodide and anti-BrdU antibody and analyzed by flow cytometry. d, CDK2-knockout MCF10A cells were arrested in G0 (Starved) and stimulated (as in c), in the presence of vehicle (Control), XL-413, CDK1 inhibitor (Ro-3306), or both inhibitors and analyzed as in c. e–g, CDK2-knockout human mammary epithelial HMEC cells. e, Whole cell lysates from parental and CDK2-knockout (sgCDK2) HMEC cells were immunoblotted as in a. f, Asynchronously growing CDK2^+/+ and CDK2-knockout HMEC cells were analyzed as in b. g, CDK2-knockout HMEC cells were arrested in G0 (Starved), stimulated to re-enter cell cycle in the presence of inhibitors and analyzed as in d. h–l, Analyses of human BJ foreskin fibroblasts expressing analog-sensitive (as) CDK2 in place of the endogenous CDK2. h, Immunoblots of whole cell lysates of parental and CDK2-knockout (sgCDK2) cells expressing asCDK2 (asCDK2) probed with an anti-CDK2 antibody. asCDK2 migrates slower due to the presence of 3xFlag-tag. i, Growth curves of asCDK2 cells treated with vehicle (Control) or 3MB-PP1 (to inhibit asCDK2). j, asCDK2 BJ fibroblasts were cultured with XL-413, 3MB-PP1, or both and analyzed as in b. k, l, asCDK2 cells were arrested in G0 (Starved) and stimulated with serum in the presence of vehicle, 3MB-PP1, XL-413, or both inhibitors. Cells were analyzed as in d. m–q, Analyses of human mammary epithelial MCF10A cells expressing analog-sensitive CDK2 (asCDK2). m, Immunoblotting as in h. n, Growth curves as in i. o, Analyses of asynchronously growing cells, as in j. p, q, analyses of cell cycle re-entry as in k, l. r–v, Analyses of human mammary epithelial HMEC cells expressing analog-sensitive CDK2 (asCDK2). r, Immunoblotting; s, growth curves (as in i); t, analyses of asynchronous cells (as in j); u, v, analyses of cell cycle re-entry (as in k, l). GAPDH was used as loading control. c, d, g, i, k, l, n, p, q, s, u, v show mean values; p-values by two-sided t-test; error bars, SD. c, d, g, i, k, l, n, p, q, s, u, v, n = 3 independent replicates; panels a, b, e, f, h, j, m, o, p, r, t, show representative results (out of 2).