Figure 2.

Cell-cycle regulation of CENP-S and CENP-X gene products in HeLa cells. HeLa cells were fractionated across the cell cycle using a double-thymidine protocol. Samples were taken at intervals for quality control and analysis of CENP regulation. (a) Flow cytometry confirms synchronous progression of cells through the cell cycle following release from thymidine block. (b) Western blot analysis with cyclin B (i) and Ser10-phospho histone H3 (ii) demonstrates normal progression through mitosis and into G1 following release. RNA (iii) isolated from each cell-cycle time point was analysed by agarose gel electrophoresis. (c) Quantitative reverse-transcript PCR was used to examine the relative abundance of control genes cyclin A, cyclin B and histone H2A (i), documenting normal cell-cycle regulation of these transcripts. CENP-S and CENP-X transcripts (ii) showed no significant modulation during the cell cycle. (d) Western blot analysis of cell cycle fractions (i) shows that CENP-S and CENP-X are uncoordinated in abundance across the cell cycle, with CENP-S maximal in G2 in parallel with CENP-A (ii). Statistical significance of signal at each time point was measured by one way analysis of variance (ANOVA) followed by a Dunnett's multiple comparison versus the 4 h time point; *p ≤ 0.01, ***p ≤ 0.001.