Figure 6: Transcriptional analysis of condensin I/CAP-H KO cells.

(a) Immunoblotting of CAP-H in CAP-H knockout and wild-type chicken DT40 cells with and without doxycycline (dox). RNAs extracted from both CAP-H^ON and CAP-H^OFF interphase synchronized cells using 14 h thymidine block were subjected to qRT–PCR analysis. Total time in dox for CAP-H^OFF (including 14 h thymidine block) was 36 h. Immunoblotting analysis shows CAP-H is completely shut down in the knockout cells, whereas the expression of CAP-H in the wild type was not affected. This is the CAP-H depletion method applied in the qRT–PCR analysis. (b) Experimental design for the analysis, showing that CAP-H KO was dox-treated up to 36 h including 14 h thymidine block. Flow cytometry profiles show that the majority of CAP-H KO cells are in G1 phase after the thymidine block. Cells were stained in propidium iodide and analysed by FACS. The CAP-H^OFF cells showed virtually no observable segregation defect at harvesting (post 36 h of dox treat)³⁸. (c) qRT–PCR analysis of CAP-H KO cells. A total of 17 genes including one tRNA gene and six histone genes were selected for analysis based on high condensin I enrichment from our genome-wide ChAP-seq data. CAP-H transcription is also shown in this graph. Overall these genes are downregulated following CAP-H removal. The error bars indicate standard error (n=3).