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. 2020 Jul 9;11:1623. doi: 10.3389/fmicb.2020.01623

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Copyright © 2020 Li, Liu, Hao, Jia, Zhao, Xie, Dong and Zeng.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cip1 acts as a dual suppressor of Cln3-Cdk1 and Ccr4-Caf120 to inhibit Whi5 function at START. (A) 10-fold serial dilutions from cultures of wild-type (YYJ138), ccr4-AID (YLP102), GAL:CIP1 (YLP105), and GAL:CIP1 ccr4-AID (YLP106) cells were spotted onto YPD and YPG with or without 1.0 mM NAA. The auxin-inducible degron (AID) system was turned on by addition of auxin (NAA) in the presence of galactose. (B) 10-fold serial dilutions from cultures of wild-type (YYJ138), ccr4-AID (YLP102), cln3Δ (YLP107), and ccr4-AID cln3Δ (YLP108) cells were spotted onto YPG with or without 1.0 mM NAA. (C) The same strains were grown to mid-log phase in YPR. Galactose was added for 1 h, and samples were taken at the indicated time points after addition of 0.5 μM NAA. Cell morphology was visualized by microscopy (scale bar, 20 μm). (D) Growth curves of the indicated strains. (E) Flow cytometry analysis of the cell volume distributions for the indicated strains (20,000 cells analyzed/sample). (F) Budded cells were counted and expressed as a percentage of total cells. (G) Deletion of WHI5 suppresses the cell-growth defect caused by Cip1 overexpression. 10-fold serial dilutions from cultures of wild-type (YFL3), whi5Δ (YLP23), GAL:CIP1 (YFL97), and GAL:CIP1 whi5Δ (YLP75) cells were spotted onto YPD and YPG medium and incubated at 30°C for 3 days. (H) The same strains were grown to mid-log phase at 30°C and then shifted to YPG medium for 18 h. Samples were collected, and cell morphology was visualized by microscopy (scale bar, 20 μm). (I) Growth curves of the indicated strains. The indicated strains were grown in YPR. Galactose (2%) was added to induce overexpression of Cip1, and growth curves were generated. (J) Budded cells were counted and expressed as a percentage of total cells. (K) Flow cytometry analysis of the cell volume distributions for the indicated strains (20,000 cells analyzed/sample). (L) 10-fold serial dilutions from cultures of wild-type (YFL3), GAL:CIP1 (YFL97), GAL:CIP1 cln3Δ (YXQ11), GAL:CIP1 whi5Δ (YLP75), and GAL:CIP1 whi5Δ cln3Δ (YLP78) cells were spotted onto YPD and YPG medium and incubated at 30°C for 3 days. (M) 10-fold serial dilutions from cultures of wild-type (YFL3), GAL:CIP1 (YFL97), GAL:CIP1 GAL:CLN3 (YLP174), and GAL:CIP1 GAL:CAF120 (YLP175) cells were spotted onto YPD and YPG medium and incubated at 30°C for 3 days. (N) Western blot analysis of Cip1 protein expression under galactose induction in the indicated strains grown in YPG.