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

Fig. 5.

Fig. 5.

The synthetic lethality between set2Δ and wee1-50 results from dNTP depletion. (A) set2Δ wee1-50 cells are sensitive to low levels of HU. WT, set2Δ, wee1-50 and set2Δ wee1-50 cells were serially diluted and spotted onto YES plates containing 5 mM HU and incubated at the permissive temperature (25°C) for 3–4 days. (B) Quantification (mean±s.e.m.) of the viability of WT and set2Δ wee1-50 cells on YES plates at 25°C containing different concentrations of HU as indicated (n≥2 experiments, n≥500 cells for each data point). **P<0.01 (t-test). (C) The endogenous protein levels of Cdc22 were examined in wee1-50 and set2Δ wee1-50 cells following incubation at 36°C for 4 h. Samples of cells were taken at the indicated time points and cell extracts were made by using the TCA method. Cdc22 was detected using an antibody against the CFP tag. α-tubulin is shown as a loading control. The lower panel shows a quantification of Cdc22 levels in wee1-50 and set2Δ wee1-50 cells for the representative blot in the upper panel. (D) dNTP levels were measured in WT, wee1-50, set2Δ and set2Δ wee1-50 strains. Cells were grown to log phase at 25°C followed by a 5 h incubation at 36°C. Samples of cells were collected and re-suspended in 10% TCA for subsequent HPLC analysis following neutralization. Means±s.e.m. of three experiments are shown. *P<0.05, **P<0.01 (t-test). (E) spd1Δ suppresses the synthetic lethality of set2Δ wee1-50. Strains were serially diluted and spotted onto YES plates and incubated at the indicated temperatures for 2–3 days. (F) dNTP levels were measured in set2Δ wee1-50 and spd1Δ set2Δ wee1-50 strains. Cells were grown to log phase at 25°C followed by a 5 h incubation at 36°C. Samples of cells were collected and re-suspended in 10% TCA for subsequent HPLC analysis following neutralization. The mean±s.e.m. for three experiments are shown. **P<0.01 (t-test between set2Δ wee1-50 and spd1Δ set2Δ wee1-50 strains; P-values: dCTP=0.3288, dGTP=0.0065, dTTP=0.0042, dATP=0.0011). The data presented are from three independent biological repeats.