RNAi depletion of ALPH1 causes growth arrest and increase in mRNA levels.

RNAi depletion of TbALPH1 was induced by tetracycline (TET). Three independent clonal cell lines were analysed over a time-course of RNAi induction. (A) Reduction in the number of ALPH1 mRNA molecules. ALPH1 mRNA and DBP1 mRNA (control) were visualized by dual colour single molecule mRNA FISH (Affymetrix), using green (ALPH1) and red (DBP1) fluorescent Affymetrix probe sets. The number of mRNA molecules per cell was quantified after 0, 24 and 48 hours of ALPH1 RNAi induction. Data are presented as box plot (waist is median; box is IQR; whiskers are ±1.5 IQR; only the smallest and largest outliers are shown; n = 100 for each time-point); the number of mRNAs from the individual cells is also presented as green (ALPH1) or red (DBP1) dots. One representative cell for each time-point is shown. The data are from one RNAi clone; data of a second clone are shown in S12 Fig. (B) Growth arrest. Growth was measured over a time-course of ALPH1 RNAi induction (±TET). Averages of the three clonal cell lines are shown; error bars indicate standard deviations between the three cell lines. (C-E) Increase in mRNAs. Total RNA was isolated over a time-course of ALPH1 RNAi and as a control from bloodstream form trypanosomes (BSF) and analysed by northern blots. The blots were probed for total mRNA with an oligo antisense to the miniexon of the spliced leader RNA (C), for PGKC (D) and for GPI-PLC (E). mRNA abundances were quantified by Odyssey (total mRNA) or phosphorimager (PGKC, GPI-PLC). rRNA was used as a loading control and all samples were calibrated to the amount of BSF RNA (= 1). Average values of the three clones are shown, standard deviations are presented as error bars. For each probe, one representative northern blot is shown. Note that the three red bands in C) are not rRNA bands, but are caused by a squeezing of the mRNAs due to the very abundant rRNA.