Suppression of DNA replication after LRR1 loss is not due to DNA damage signaling (related to Fig. 3). (A) Effect of LRR1 knockout was measured in p53−/− cells. Rate of EdU incorporation in S-phase cells was normalized to control cells. Two-sample Student’s t test; P = 6.0 × 10−4 (n = 2 technical replicates, n ≥ 1,864 cells per replicate). Data are representative of two independent experiments. (B) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs, released from starvation 2 d later, and fixed after 30 h. Left: Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Two-sample Student’s t test; P = 1.8 × 10−4, 2.4 × 10−3 (n = 2 technical replicates, n ≥ 1,904 cells per replicate). Right: Nuclear abundance of p21 in S-phase cells normalized to control cells. Two-sample Student’s t test; P = 3.2 × 10−3, 1.7 × 10−3 (n = 2 technical replicates, n ≥ 1,904 cells per replicate). It was previously reported that CRL2LRR1 targets the CDK inhibitor p21 for degradation (Starostina et al., 2010). While LRR1 knockout resulted in higher levels of nuclear p21 in S phase, knockdown of p21 did not rescue the DNA replication defect after LRR1 knockout. (C) Rate of EdU incorporation in S-phase cells after 4-h treatment with ATM inhibitor (ATMi; KU-60019, 5 µM), DNA-PK inhibitor (DNA-PKi; NU7441, 1 µM), ATR inhibitor (ATRi; AZ20, 1 µM), Chk1 inhibitor (Chk1i; CHIR124, 500 nM), or Wee1 inhibitor (Wee1i; MK1775, 1 µM) and normalized to control cells (n ≥ 2,909 cells per condition). Data from n = 2 independent experiments. (D) 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 negative control, the same cell line was treated with doxycycline and nontargeting siRNA. As a positive control, cells were treated with 10 µM aphidicolin for 3 h. The indicated protein markers were measured in S-phase 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 ≥ 959 cells per condition). The lack of increase in damage signal was not due to measurement insensitivity, as positive control cells treated with the DNA polymerase inhibitor aphidicolin showed elevated signals, and more importantly, elevated signals were also detected when these LRR1-null cells progressed into G2 phase (Fig. 4 I). In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure S3.

Suppression of DNA replication after LRR1 loss is not due to DNA damage signaling (related to Fig. 3). (A) Effect of LRR1 knockout was measured in p53−/− cells. Rate of EdU incorporation in S-phase cells was normalized to control cells. Two-sample Student’s t test; P = 6.0 × 10−4 (n = 2 technical replicates, n ≥ 1,864 cells per replicate). Data are representative of two independent experiments. (B) Serum-starved cells stably expressing Cas9 were transfected with sgRNAs, released from starvation 2 d later, and fixed after 30 h. Left: Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Two-sample Student’s t test; P = 1.8 × 10−4, 2.4 × 10−3 (n = 2 technical replicates, n ≥ 1,904 cells per replicate). Right: Nuclear abundance of p21 in S-phase cells normalized to control cells. Two-sample Student’s t test; P = 3.2 × 10−3, 1.7 × 10−3 (n = 2 technical replicates, n ≥ 1,904 cells per replicate). It was previously reported that CRL2LRR1 targets the CDK inhibitor p21 for degradation (Starostina et al., 2010). While LRR1 knockout resulted in higher levels of nuclear p21 in S phase, knockdown of p21 did not rescue the DNA replication defect after LRR1 knockout. (C) Rate of EdU incorporation in S-phase cells after 4-h treatment with ATM inhibitor (ATMi; KU-60019, 5 µM), DNA-PK inhibitor (DNA-PKi; NU7441, 1 µM), ATR inhibitor (ATRi; AZ20, 1 µM), Chk1 inhibitor (Chk1i; CHIR124, 500 nM), or Wee1 inhibitor (Wee1i; MK1775, 1 µM) and normalized to control cells (n ≥ 2,909 cells per condition). Data from n = 2 independent experiments. (D) 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 negative control, the same cell line was treated with doxycycline and nontargeting siRNA. As a positive control, cells were treated with 10 µM aphidicolin for 3 h. The indicated protein markers were measured in S-phase 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 ≥ 959 cells per condition). The lack of increase in damage signal was not due to measurement insensitivity, as positive control cells treated with the DNA polymerase inhibitor aphidicolin showed elevated signals, and more importantly, elevated signals were also detected when these LRR1-null cells progressed into G2 phase (Fig. 4 I). In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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