Persistent binding of CMG helicases to chromatin after LRR1 loss blocks mitosis by activating ATR–Chk1–Wee1. (A) Serum-starved Cas9 cells were transfected with sgRNAs, released from starvation, and fixed after 30 h. Sample immunofluorescence images are shown. Arrowheads indicate cells with positive phospho-H3-Ser10 signal. Scale bar: 20 µm. Data are representative of three independent experiments. (B) Schematic of gating for G2/M-phase cells (n = 3,000 random cells displayed). (C) Histogram of phospho-H3-Ser10 signal in G2/M cells (cutoff = 11, n ≥ 4,389 cells per condition). (D) Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 0.048 (n = 3 independent experiments, n ≥ 4,389 cells per condition). Right: Abundance of chromatin-bound CDC45 in G2/M-phase cells normalized to control cells. Paired Student’s t test; P = 0.021 (n = 3 independent experiments, n ≥ 4,389 cells per condition). (E) Continuously cycling Cas9 cells were transfected with sgRNAs and fixed 1 d later, as in F–H. Percentage of G2/M cells with positive phospho-H3-Ser10 signal normalized to control cells (sgCNTL + DMSO). Paired Student’s t test, columns 1 vs. 3: P = 0.027; columns 3 vs. 4: P = 6.9 × 10−3; columns 2 vs. 4: P = 0.20 (n = 2 independent experiments, n ≥ 4,132 cells per condition). (F) Scatter plot of phospho-H3-Ser10 signal against abundance of chromatin-bound CDC45 in G2/M-phase cells (n = 3,000 random cells displayed). Data are representative of three independent experiments. (G) Percentage of G2/M cells with positive phospho-H3-Ser10 signal was normalized to control cells. Cells were categorized as CDC45low or CDC45high based on the abundance of chromatin-bound CDC45 (see Fig. 4 F, light- and dark-shaded areas). Paired Student’s t test; P = 6.3 × 10−3, 8.4 × 10−5 (n = 4 independent experiments, n ≥ 1,322 cells per condition). (H) Cells were treated with the ATR inhibitor (ATRi; AZ20, 1 µM), Chk1 inhibitor (Chk1i; CHIR124, 500 nM), or Wee1 inhibitor (Wee1i; MK1775, 1 µM) for 4 h. Percentage of G2/M cells with positive phospho-H3-Ser10 signal was normalized to control cells (sgCNTL + DMSO). Paired Student’s t test; P = 0.041, 0.012, 0.037, 0.011 (n = 3 independent experiments, n ≥ 2,804 cells per condition). (I) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1. Positive control cells were treated with 10 µM aphidicolin for 3 h. The indicated protein markers were measured in G2/M cells through immunofluorescence, and nuclear signals were normalized to control cells (LRR1 positive). Paired Student’s t test; *, P < 0.05; **, P < 0.01; *** P < 0.001 (n = 3 independent experiments, n ≥ 4,000 cells per condition). A.U., arbitrary unit; norm., normalized; R.F.U., relative fluorescence unit; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4.

Persistent binding of CMG helicases to chromatin after LRR1 loss blocks mitosis by activating ATR–Chk1–Wee1. (A) Serum-starved Cas9 cells were transfected with sgRNAs, released from starvation, and fixed after 30 h. Sample immunofluorescence images are shown. Arrowheads indicate cells with positive phospho-H3-Ser10 signal. Scale bar: 20 µm. Data are representative of three independent experiments. (B) Schematic of gating for G2/M-phase cells (n = 3,000 random cells displayed). (C) Histogram of phospho-H3-Ser10 signal in G2/M cells (cutoff = 11, n ≥ 4,389 cells per condition). (D) Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 0.048 (n = 3 independent experiments, n ≥ 4,389 cells per condition). Right: Abundance of chromatin-bound CDC45 in G2/M-phase cells normalized to control cells. Paired Student’s t test; P = 0.021 (n = 3 independent experiments, n ≥ 4,389 cells per condition). (E) Continuously cycling Cas9 cells were transfected with sgRNAs and fixed 1 d later, as in F–H. Percentage of G2/M cells with positive phospho-H3-Ser10 signal normalized to control cells (sgCNTL + DMSO). Paired Student’s t test, columns 1 vs. 3: P = 0.027; columns 3 vs. 4: P = 6.9 × 10−3; columns 2 vs. 4: P = 0.20 (n = 2 independent experiments, n ≥ 4,132 cells per condition). (F) Scatter plot of phospho-H3-Ser10 signal against abundance of chromatin-bound CDC45 in G2/M-phase cells (n = 3,000 random cells displayed). Data are representative of three independent experiments. (G) Percentage of G2/M cells with positive phospho-H3-Ser10 signal was normalized to control cells. Cells were categorized as CDC45low or CDC45high based on the abundance of chromatin-bound CDC45 (see Fig. 4 F, light- and dark-shaded areas). Paired Student’s t test; P = 6.3 × 10−3, 8.4 × 10−5 (n = 4 independent experiments, n ≥ 1,322 cells per condition). (H) Cells were treated with the ATR inhibitor (ATRi; AZ20, 1 µM), Chk1 inhibitor (Chk1i; CHIR124, 500 nM), or Wee1 inhibitor (Wee1i; MK1775, 1 µM) for 4 h. Percentage of G2/M cells with positive phospho-H3-Ser10 signal was normalized to control cells (sgCNTL + DMSO). Paired Student’s t test; P = 0.041, 0.012, 0.037, 0.011 (n = 3 independent experiments, n ≥ 2,804 cells per condition). (I) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1. Positive control cells were treated with 10 µM aphidicolin for 3 h. The indicated protein markers were measured in G2/M cells through immunofluorescence, and nuclear signals were normalized to control cells (LRR1 positive). Paired Student’s t test; *, P < 0.05; **, P < 0.01; *** P < 0.001 (n = 3 independent experiments, n ≥ 4,000 cells per condition). A.U., arbitrary unit; norm., normalized; R.F.U., relative fluorescence unit; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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