Fig 4. Down regulation of CYC4 causes S phase defects in HAT2-depleted cells.
a. Analysis of differential expression of S phase cyclins in LdHAT2-hKO:hyg versus Ld1S cells by real time PCR analysis of RNA using 2-ΔΔCT method (in which tubulin served as internal control). Two-tailed student’s t-test was applied: **p < 0.005. b. Western blot analysis of whole cell lysates isolated from Ld1S-hyg cells expressing either eGFP or CYC4-eGFP and LdHAT2-hKO cells expressing CYC4-eGFP (2x108 cell equivalents per well) using anti-eGFP antibodies (1:2000 dilution). 1/10 of each sample was loaded for tubulin control. c. Growth analysis of LdHAT2-hKO:hyg cells expressing CYC4-eGFP ectopically in comparison with LdHAT2-hKO:hyg/HAT2-eGFP rescue line, LdHAT2-hKO:hyg/eGFP heterozygous knockout line, control line Ld1S-hyg/eGFP and Ld1S-hyg cells expressing CYC4-eGFP ectopically. Three separate experiments were initiated in parallel. Values plotted are the average of three experiments, error bars represent standard deviation. d. Flow cytometry analysis of HU-synchronized LdHAT2-hKO cells expressing CYC4-eGFP ectopically in comparison with LdHAT2-hKO cells expressing eGFP and control line Ld1S-hyg/eGFP. Time after release at which sampling was done is indicated above each column of histograms. 30,000 events were analyzed at every time-point, and M1, M2 and M3 gates indicate G1, S and G2/M phases. The percent of cells at different cell cycle stages are indicated in the insets of each histogram. Data set of one of the three experiments performed is shown. e. Flow cytometry analysis of flavopiridol-synchronized LdHAT2-hKO cells expressing CYC4-eGFP ectopically in comparison with LdHAT2-hKO cells expressing eGFP, LdHAT2-hKO:hyg/HAT2-eGFP rescue line, and control line Ld1S-hyg/eGFP. Data set of one of the three experiments performed is shown.