Figure 5.

Knockdown of TbSCC1 results in an increase in the in situ switching to VSG121 ES. (A) RNAi-mediated knockdown of different subunits of the cohesin complex was induced over 4 d in bloodstream form trypanosomes. Indirect IF using anti-VSG221 or anti-VSG121 antibodies and DAPI staining was performed. (A and B) The frequency of cells negative for VSG221 labeling (A) or positive for VSG121 labeling (B) is shown. Mean ± SEM of experiments (n > 500) for two independent clones is shown for each gene. (C and D) Double indirect IF with rabbit anti-VSG221 (green), mouse anti-VSG121 (red), and DAPI staining (blue) was performed upon TbSCC1 knockdown. Representative pictures show a trypanosome positive only for VSG121 labeling (C; arrowheads) or positive for both VSGs (D; arrowheads). Yellow indicates double labeling. (E–G) Knockdown of TbSCC1 promotes the appearance of VSG121-expressing variants that are able to proceed through the cell cycle. Representative pictures showing bloodstream trypanosomes positive only for VSG121 labeling and displaying different nucleus/kinetoplast numbers are shown: 1K1N (E), 2K1N (F), and 2K2N (G). Arrowheads indicate the position of the cell in which expression of the VSG gene has changed from VSG221 to VSG121. Bars, 5 µm.