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. 2014 Jan 6;1(1):9–20. doi: 10.15698/mic2014.01.120

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(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 10⁴ml^-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 22) 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.