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Fig. 7. Overexpression of DmCdc6 in S2 cells has only a small effect on the cells. (A) FACS profile of cells in which DmCdc6 has been overexpressed. (B) Overexpressed Dm Cdc6 is nuclear: S2 Cdc6-1 and S2 Cdc6-2 cells were fractionated and analysed by western blotting. 500,000 equivalent cells were loaded per lane. T, total cell extract; C, cytoplasmic extract; Np, nucleoplasmic extract; Chr, chromatin-bound extract. (C) Immunofluorescent staining of S2 Cdc6-1 and S2 Cdc6-2 cell lines using the antibody directed against the His tag on the protein. The His staining is shown in green and the DNA, which is stained with DAPI, is shown in blue. (D) RTPCR to show the effects of the depletion of the endogenous DmCdc6 on S2 cells overexpressing DmCdc6. Normal S2, S2 Cdc6-1 and S2 Cdc6-2 cells were treated with UTR RNAi (Fig. 3A) and then subject to RTPCR using either RTPCR1 or RTPCR2 to detect total and endogenous DmCdc6, respectively. ORC1 in this case serves as the negative control. + indicates RNAi treatment and – no RNAi treatment. (E) Growth analysis of normal S2, S2 Cdc6-1 and S2 Cdc6-2 cells with and without Cdc6 5' UTR RNAi treatment.
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