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. 2019 Mar 25;132(6):jcs226969. doi: 10.1242/jcs.226969

Fig. 7.

Fig. 7.

rad3Δ and chk1Δ are synthetic lethal with wee1-50 through replication stress. (A) set2Δ wee1-50, rad3Δ wee1-50 or chk1Δ wee1-50 result in premature entry into mitosis but the proportion of cells with the ‘cut’ phenotype in the set2Δ wee1-50 mutant is significantly lower at earlier time points. set2Δ wee1-50, rad3Δ wee1-50 and chk1Δ wee1-50 cells were grown to log phase at the permissive temperature (25°C), then incubated at 36°C to inactivate Wee1. Samples were fixed with 70% ethanol at the indicated times. The fixed cells were stained with DAPI and examined by microscopy analysis. (B) Quantitative analysis of cells in A. Means±s.e.m. are shown. **P<0.01 (t-test); black asterisks indicates statistically significant differences between rad3Δ wee1-50 cells grown at either 36°C or 25°C (P-values for rad3Δ wee1-50 cells: 4h=0.0017, 5h=0.0003; averages of n≥2 experiments, n≥100 cells for each data point); blue asterisks indicate statistically significant differences between chk1Δ wee1-50 cells grown at either 36°C or 25°C (P-values for chk1Δ wee1-50 cells: 4 h=0.0006, 5h=0.0006; averages of n≥2 experiments, n≥100 cells for each data point); orange asterisks indicate statistically significant differences between set2Δ wee1-50 cells grown at 36°C or 25°C (P-values for set2Δ wee1-50 cells: 4h=0.0002, 5h=0.0078; averages of n≥2 experiments, n≥100 cells for each data point). (C) Wee1 inactivation causes replication stress in rad3Δ or chk1Δ mutants. Flow cytometric analysis of WT, rad3Δ, chk1Δ, rad3Δ wee1-50 or chk1Δ wee1-50 cells at 25°C or 36°C at the indicated time points. (D) dNTP levels were measured in WT, wee1-50, rad3Δ wee1-50 and chk1Δ wee1-50 strains. These strains were grown to log phase at 25°C following by a 5 h incubation at 36°C. Samples of cells were collected and re-suspended in 10% TCA for HPLC analysis. Means±s.e.m. of three biological repeats are shown. *P<0.05, **P<0.01 (t-test). (E) Total dNTP levels are reduced in wee1-50, rad3Δ wee1-50 or chk1 Δ wee1-50 cells compared to WT cells. **P<0.01 (t-test; P-values: wee1-50=0.0012, rad3Δ wee1-50=0.0075, chk1Δ wee1-50=0.0059). The data presented are from three independent biological repeats. (F) spd1Δ suppresses the synthetic lethality of chk1Δ wee1-50. Strains were serially diluted and spotted onto YES plates and incubated at indicated temperatures for 2–3 days. (G) spd1Δ cannot suppress the synthetic lethality of rad3Δ wee1-50. A similar experiment was carried out as described in F.