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. 2016 Jul 27;12(7):e1006216. doi: 10.1371/journal.pgen.1006216

Fig 4. Constitutive expression of Ycg1 delays progression through the cell cycle.

Fig 4

(A) Doubling time of strains expressing the indicated Ycg1 proteins from the endogenous locus (YTD33, YTD200, YTD148, YTD201, YTD164). Mean doubling time from 3 independent experiments, +/- 1 standard deviation, are shown. (B) Strains expressing 3HA-tagged Ycg1 (YTD33) or Ycg1-K977A (YTD148) from the endogenous locus were arrested in G1 with alpha-factor for 3 hours and then released into the cell cycle. Alpha-factor was added back after 45 minutes to prevent cells from entering a second cycle. Western blots of Ycg1-3HA, Clb2 and Cdk1 are shown (top). Flow cytometry plots (bottom) illustrate the delayed progression of ycg1-K977A cells into S phase (compare 30 and 45-minute time points). (C) Doubling time of wild-type (YTD33) and ycg1-K977A strains (YTD148) were compared to strains overexpressing each of the indicated condensin subunits from the TEF1 promoter (YTD336, YTD337, YTD353, YTD349, YTD362). Mean doubling times from 3 independent experiments are shown, +/- 1 standard deviation. (D) YCG1 (YTD276) and TEFp-YCG1 (YTD361) strains were arrested in metaphase with a MET3p-CDC20 shut-off allele for 3 hours, then released into alpha-factor for 2 hours to synchronize cells in G1. Alpha-factor was added back 45 minutes after release from G1 arrest to prevent cells from entering a second cycle. Western blots of Ycg1-3HA, Clb2 and Cdk1 are shown (top). Flow cytometry plots (bottom) demonstrate the delayed progression of ycg1-K977A cells into S phase (compare 30 and 45-minute time points).