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. 2022 Jul 7;16(15):2771–2787. doi: 10.1002/1878-0261.13264

Fig. 4.

Fig. 4

Accumulation of p27 provides a survival advantage in the presence of CHK1i. (A) Western blot showing SKP2 and p27 protein levels in SKP2 KO and control clones. A single experiment was performed. (B) Baf3 SKP2 KO and sg‐Cd8a control clones were seeded at a concentration of 3.5 × 105 cells per well. Cells were split 1 : 1 and counted every 24 h over the course of 4 days using an Attune NxT Flow cytometer. Line graph data points represent the mean (±SD) of three technical replicates per time point and three individual experiments per clone. (C) Baf3 SKP2 KO and sg‐Cd8a control clones (dsRed+) were mixed 1 : 1 ratio with Baf3Cas9 cells (parental cells) and the percentage of dsRed+ cells was monitored over time using flow cytometry. Each line represents the mean (±SD) of dsRed+ (%) cells normalized to the 48 h timepoint in three independent experiments per clone, except sg‐Cd8a‐1cl1 and sg‐Cd8a‐2cl2 (n = 2). (D) Baf3 SKP2 KO and sg‐Cd8a control clones (dsRed+) were mixed at a ratio of 15/85 with parental Baf3Cas9 cells and treated with low doses of the CHK1 inhibitor CHIR‐124 for up to 7 days. The percentage of dsRed+ cells was assessed using flow cytometry, whereby the time points were normalized to 0 h. Cells were split every 2–3 days. Lines represent mean ratio (±SEM) of three independent experiments. (E) Representative contour plots of EdU cell cycle analysis of a Baf3 SKP2 KO and sg‐Cd8a control clone. Three independent experiments were performed. (F) Quantification of cell cycle analysis of Baf3 SKP2 KO and sg‐Cd8a control clones using EdU‐staining. Bars indicate the mean percentage (±SD) of two independent single‐cell clones per genotype analyzed in three independent experiments. (G) Representative contour plots of EdU cell cycle analysis of Baf3 SKP2 KO and sg‐Cd8a control clones after 24 h of PF‐477736 treatment. Three independent experiments were performed. (H) Quantification of cell cycle analysis of Baf3 SKP2 KO and sg‐Cd8a control clones using EdU‐staining after 24 h of PF‐477736 treatment. Bars indicate the mean percentage (±SD) of two different single‐cell clones per genotype analyzed in three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; unpaired t‐test.