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. 2021 Jul 14;17(7):e1009560. doi: 10.1371/journal.pgen.1009560

Fig 5. The largely cytoplasmic GFP-NES-Pch2 version supports checkpoint function.

Fig 5

(A) Time course analysis of meiotic nuclear divisions; the percentage of cells containing two or more nuclei is represented. Error bars: SD; n = 3. At least 300 cells were scored for each strain at every time point. (B) Spore viability assessed by tetrad dissection is shown in the top graph. The percentage of tetrads containing 4-, 3-, 2-, 1-, and 0-viable spores is presented in the bottom graph. At least 54 tetrads were dissected for each strain. Error bars, SD; n = 3. (C) Representative western blot analysis of GFP-Pch2 and Hop1 production, and Hop1-T318 and H3-T11 phosphorylation (ph), in ndt80Δ-arrested strains of the indicated genotypes. Cell extracts were prepared at 24 hours in meiosis. (D) Quantification of GFP-Pch2, Hop1, Hop1-T318ph and H3-T11ph relative levels analyzed as in (C). The intensity values for each protein in each strain were normalized to Pgk1 and relativized to those of the wild-type GFP-PCH2 strain. Error bars, SD; n = 3. Light grey and dark grey bars correspond to ZIP1 and zip1Δ strains, respectively. Strains in (A) are: DP1624 (GFP-PCH2), DP1685 (GFP-NES-PCH2), DP1699 (GFP-NLS-PCH2), DP1695 (GFP-NLS-PCH2 [hom]), DP1023 (pch2Δ), DP1625 (zip1Δ GFP-PCH2), DP1686 (zip1Δ GFP-NES-PCH2), DP1701 (zip1Δ GFP-NLS-PCH2), DP1696 (zip1Δ GFP-NLS-PCH2 [hom]) and DP1029 (zip1Δ pch2Δ). Strains in (B) are: DP1624 (GFP-PCH2), DP1023 (pch2Δ), DP1685 (GFP-NES-PCH2), DP1699 (GFP-NLS-PCH2), DP1695 (GFP-NLS-PCH2 [hom]), DP1789 (spo11-3HA GFP-PCH2), DP1787 (spo11-3HA pch2Δ), DP1791 (spo11-3HA GFP-NES-PCH2), DP1793 (spo11-3HA GFP-NLS-PCH2) and DP1792 (spo11-3HA GFP-NLS-PCH2 [hom]). Strains in (C, D) are: DP1654 (ndt80Δ GFP-PCH2), DP1725 (ndt80Δ GFP-NES-PCH2), DP1729 (ndt80Δ GFP-NLS-PCH2), DP1768 (ndt80Δ GFP-NLS-PCH2 [hom]), DP1058 (ndt80Δ pch2Δ), DP1655 (ndt80Δ zip1Δ GFP-PCH2), DP1726 (ndt80Δ zip1Δ GFP-NES-PCH2), DP1730 (ndt80Δ zip1Δ GFP-NLS-PCH2), DP1769 (ndt80Δ zip1Δ GFP-NLS-PCH2 [hom]) and DP881 (ndt80Δ zip1Δ pch2Δ).