FIGURE 6: RNAi of TOR genes in bloodstream form.

(A) Growth analysis of BSF RNAi mutants in vitro. RNAi of TOR genes in BSF 2T1 constitutively expressing YFP-ATG8.1 (diamonds) and YFP-ATG8.2 (squares). RNAi induced with tetracycline (filled symbols) and growth compared to controls (empty symbols) for 96 h. Cells were reseeded to 1 x 10⁴ ml^-1 at 48 h as required, with cumulative growth shown. Arrows indicate time point selected for microscopy analysis following. Inset: confirmation of RNAi specificity by western blot (where antibody available, TOR1 [1]) or by qPCR of TOR RNAi cell lines at selected analysis time points after tetracycline induction (hatch) or control in BSF 2T1 clones expressing either YFP-ATG8.1 (grey) or YFP-ATG8.2 (black). Error bars represent one standard deviation derived from three replicates.

(B) The mean number of autophagosomes per cell was determined in TOR RNAi lines by counting >200 cells at selected times after induction (arrows in A), with data displayed as a mean of at least three replicate experiments. Error bars represent standard deviation and asterisks indicate where data differed significantly from the mean of the un-induced controls *p<0.05. Constitutive expression of YFP-ATG8.2 in BSF 2T1 cells was visualised by fluorescent microscopy following individual RNAi of TOR1. Left hand images FITC filter set, right hand images FITC DIC merge. Scale bar 5 µm.

References

1. A. Barquilla, J.L. Crespo, and M. Navarro, "Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation", Proceedings of the National Academy of Sciences, vol. 105, pp. 14579-14584, 2008. http://dx.doi.org/10.1073/pnas.0802668105