JCS02567 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. Rhizoxin does not reverse the G1-S block in CRK1+CRK2-deficient procyclic-form T. brucei. Rhizoxin (1 nM) was added to the CRK1+CRK2-deficient procyclic-form cells at the same time as tetracycline for RNAi induction. The cells were cultured for 3 days. BrdU was then added and the incubation was continued for a further 2 days. Cells were collected and examined in the immunofluorescence assay. Top panel, control cells. Middle panel, CRK1+CRK2-depleted cells. Lower panel, Rhizoxin-treated CRK1+CRK2-depleted cells. Note that BrdU is not incorporated into the nuclei of either the CRK1+CRK2-deficient or rhizoxin-treated CRK1+CRK2-deficient cells.

• Supplemental Figure 2 - Fig. S2. Rhizoxin does not affect the RNAi effects in knocking down the expression of CRK1 and CRK2 in procyclic-form T. brucei. Rhizoxin (1 nM) was added to the procyclic-form trypanosome cells harboring the CRK1+CRK2 RNAi plasmid construct at the same time when RNAi was induced. Cells were collected after a 3-day incubation and semi-quantitative RT-PCR was performed using the RNA extracted from the cells as template. a-Tubulin mRNA (TUB) was included as a sampling control.

• Supplemental Figure 3 - Fig. S3. The in vitro differentiation of CRK1+CRK2-deficient and CycE1/CYC2+CRK1+CRK2-deficient bloodstream-form cells into the procyclic form. Cells with RNAi induced for 3 days were initiated for in vitro differentiation. After 60 hours of differentiation, cells were collected, stained with DAPI and examined with a fluorescence microscope. Top panel, bloodstream-form cells; middle panel, differentiated CRK1+CRK2 double knockdown procyclic-form cells; lower panel, differentiated CRK1+CRK2+CycE1/CYC2 triple knockdown procyclic-form cells. Note that the positions of kinetoplasts in the newly differentiated procyclic form have moved midway between the nuclei and posterior end.