Analysis of the G2/M checkpoint and under-replicated DNA after LRR1 loss (related to Fig. 4). (A) Continuously cycling cells stably expressing Cas9 were transfected with sgRNAs and fixed 1 d later. Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 3.3 × 10−3 (n = 4 independent experiments, n ≥ 3,421 cells per condition). Right: Abundance of chromatin-bound CDC45 in G2/M-phase cells normalized to control cells. Paired Student’s t test; P = 1.3 × 10−3 (n = 4 independent experiments, n ≥ 3,421 cells per condition). (B) Cells were treated with DMSO or a p97 inhibitor (CB-5083, 4 µM) for 6 h. Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 0.044 (n = 2 independent experiments, n ≥ 3,522 cells per condition). (C) Top: Schematic of cyclin E/A–CDK activity reporter. DHB is a fragment (aa 994–1087) of the human DHB protein. The reporter translocates to the cytoplasm when phosphorylated by cyclin E/A–CDK activity. Bottom: Cells expressing H2B-mTurquoise and the cyclin E/A–CDK activity reporter were released from serum starvation and imaged and further categorized as CDK-high if cyclin E/A–CDK activity rose above 0.85 for more than 1 h. Mitosis was monitored by the separation of condensed chromatin during anaphase (see bottom right sample images). Cells #1 and #2 underwent mitosis at 22.5 h and 25 h after release, respectively. Cells #3 and #4 did not undergo mitosis within 30 h after release. Scale bar: 10 µm. (D) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs and released from starvation 2 d later. Cyclin E/A–CDK activity and mitotic time were measured (n = 50 sample traces displayed). Data are representative of three independent experiments. (E) Quantification of D. Percentage of CDKhigh cells that underwent mitosis is plotted as a function of time since serum release (n = 6,016 and 5,979 cells). Fisher’s exact test; ***, P < 1 × 10−3. Data are representative of three independent experiments. (F) Cells expressing CRISPR-resistant LRR1 from a doxycycline-inducible promoter were transfected with sgCNTL or sgLRR1 and selected for stable single-cell clones. Cells were cultured in doxycycline to maintain expression of exogenous LRR1. Percentage of G2/M cells with positive phospho-H3-Ser10 signal was measured after replacing doxycycline with DMSO or doxycycline for 1 d. Two-sample Student’s t test; P = 0.38, 0.016, 3.7 × 10−3 (n = 2 technical replicates, n ≥ 2,104 cells per replicate). (G) Effect of LRR1 knockout was measured in p53−/− cells. Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 0.014 (n = 2 technical replicates, n ≥ 4,111 cells per replicate). (H) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs, released from starvation 2 d later, and fixed after 30 h. Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 1.1 × 10−3, 0.11 (n = 2 technical replicates, n ≥ 5,807 cells per replicate). Right: Nuclear abundance of p21 in G2/M cells normalized to control cells (sgCNTL + siCNTL). Two-sample Student’s t test; P = 8.7 × 10−4, 1.0 × 10−4 (n = 2 technical replicates, n ≥ 5,807 cells per replicate). (I) Percentage of G2/M cells with positive phospho-H3-Ser10 signal after 4-h treatment with ATM inhibitor (ATMi; KU-60019, 5 µM), DNA-PK inhibitor (DNA-PKi; NU7441, 1 µM), or ATR inhibitor (ATRi; AZ20, 1 µM; n ≥ 4,648 cells per condition). Data from two independent experiments. (J) Histogram of chromatin-bound CDC45 in G1-phase daughter cells or G2/M cells after 4-h treatment with DMSO or Wee1 inhibitor (Wee1i; MK1775, 1 µM; n ≥ 356 cells per condition). Data are representative of two technical replicates. (K) Sample immunofluorescence images from experiment in Fig. 4 I. Scale bar: 20 µm. Data are representative of three independent experiments. (L) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1 by removing doxycycline and transfecting siRNA targeting exogenous LRR1 1 d before fixation. As a positive control, cells were treated with the indicated concentration of aphidicolin for 16 h. Top: Cells were treated with Wee1 inhibitor (MK1775, 1 µM) for 1 h and pulse labeled with EdU for 10 min. EdU puncta area was quantified in mitotic cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 7.2 × 10−4, 0.047, 0.038, 0.025, 1.5 × 10−3 (n = 2 technical replicates, n ≥ 209 cells per condition). Bottom: Cells were treated with Wee1 inhibitor (MK1775, 1 µM) for 3 h, and chromatin-bound 53BP1 puncta area was quantified in G1-phase daughter cells born after Wee1 inhibition. Two-sample Student’s t test; P = 0.0498, 0.030, 0.32, 0.77, 0.090 (n = 2 technical replicates, n ≥ 582 cells per condition). R.F.U., relative fluorescence unit; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure S5.

Analysis of the G2/M checkpoint and under-replicated DNA after LRR1 loss (related to Fig. 4). (A) Continuously cycling cells stably expressing Cas9 were transfected with sgRNAs and fixed 1 d later. Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 3.3 × 10−3 (n = 4 independent experiments, n ≥ 3,421 cells per condition). Right: Abundance of chromatin-bound CDC45 in G2/M-phase cells normalized to control cells. Paired Student’s t test; P = 1.3 × 10−3 (n = 4 independent experiments, n ≥ 3,421 cells per condition). (B) Cells were treated with DMSO or a p97 inhibitor (CB-5083, 4 µM) for 6 h. Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Paired Student’s t test; P = 0.044 (n = 2 independent experiments, n ≥ 3,522 cells per condition). (C) Top: Schematic of cyclin E/A–CDK activity reporter. DHB is a fragment (aa 994–1087) of the human DHB protein. The reporter translocates to the cytoplasm when phosphorylated by cyclin E/A–CDK activity. Bottom: Cells expressing H2B-mTurquoise and the cyclin E/A–CDK activity reporter were released from serum starvation and imaged and further categorized as CDK-high if cyclin E/A–CDK activity rose above 0.85 for more than 1 h. Mitosis was monitored by the separation of condensed chromatin during anaphase (see bottom right sample images). Cells #1 and #2 underwent mitosis at 22.5 h and 25 h after release, respectively. Cells #3 and #4 did not undergo mitosis within 30 h after release. Scale bar: 10 µm. (D) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs and released from starvation 2 d later. Cyclin E/A–CDK activity and mitotic time were measured (n = 50 sample traces displayed). Data are representative of three independent experiments. (E) Quantification of D. Percentage of CDKhigh cells that underwent mitosis is plotted as a function of time since serum release (n = 6,016 and 5,979 cells). Fisher’s exact test; ***, P < 1 × 10−3. Data are representative of three independent experiments. (F) Cells expressing CRISPR-resistant LRR1 from a doxycycline-inducible promoter were transfected with sgCNTL or sgLRR1 and selected for stable single-cell clones. Cells were cultured in doxycycline to maintain expression of exogenous LRR1. Percentage of G2/M cells with positive phospho-H3-Ser10 signal was measured after replacing doxycycline with DMSO or doxycycline for 1 d. Two-sample Student’s t test; P = 0.38, 0.016, 3.7 × 10−3 (n = 2 technical replicates, n ≥ 2,104 cells per replicate). (G) Effect of LRR1 knockout was measured in p53−/− cells. Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 0.014 (n = 2 technical replicates, n ≥ 4,111 cells per replicate). (H) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs, released from starvation 2 d later, and fixed after 30 h. Left: Percentage of G2/M cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 1.1 × 10−3, 0.11 (n = 2 technical replicates, n ≥ 5,807 cells per replicate). Right: Nuclear abundance of p21 in G2/M cells normalized to control cells (sgCNTL + siCNTL). Two-sample Student’s t test; P = 8.7 × 10−4, 1.0 × 10−4 (n = 2 technical replicates, n ≥ 5,807 cells per replicate). (I) Percentage of G2/M cells with positive phospho-H3-Ser10 signal after 4-h treatment with ATM inhibitor (ATMi; KU-60019, 5 µM), DNA-PK inhibitor (DNA-PKi; NU7441, 1 µM), or ATR inhibitor (ATRi; AZ20, 1 µM; n ≥ 4,648 cells per condition). Data from two independent experiments. (J) Histogram of chromatin-bound CDC45 in G1-phase daughter cells or G2/M cells after 4-h treatment with DMSO or Wee1 inhibitor (Wee1i; MK1775, 1 µM; n ≥ 356 cells per condition). Data are representative of two technical replicates. (K) Sample immunofluorescence images from experiment in Fig. 4 I. Scale bar: 20 µm. Data are representative of three independent experiments. (L) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1 by removing doxycycline and transfecting siRNA targeting exogenous LRR1 1 d before fixation. As a positive control, cells were treated with the indicated concentration of aphidicolin for 16 h. Top: Cells were treated with Wee1 inhibitor (MK1775, 1 µM) for 1 h and pulse labeled with EdU for 10 min. EdU puncta area was quantified in mitotic cells with positive phospho-H3-Ser10 signal. Two-sample Student’s t test; P = 7.2 × 10−4, 0.047, 0.038, 0.025, 1.5 × 10−3 (n = 2 technical replicates, n ≥ 209 cells per condition). Bottom: Cells were treated with Wee1 inhibitor (MK1775, 1 µM) for 3 h, and chromatin-bound 53BP1 puncta area was quantified in G1-phase daughter cells born after Wee1 inhibition. Two-sample Student’s t test; P = 0.0498, 0.030, 0.32, 0.77, 0.090 (n = 2 technical replicates, n ≥ 582 cells per condition). 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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