Fig. 3.

TbARL6 knockdown by RNA interference (A) Total cell lysates (5 × 10⁶ cells/lane) from BSF parental line Lister 427 (wt) and cell line 427/p2T7ARL6 (RNAi) grown in the presence of tetracycline for 0–72 h were immunoblotted and probed with anti-TbARL6 and anti-NMT to monitor equal sample loading. (B) Cumulative growth of BSF parental line Lister 427 (wt) and transfected line 427/p2T7ARL6 (RNAi) in the absence and presence of tetracycline, monitored over a 5 day time course. Mean values (± SD) are plotted (n = 3), error bars not visible. (C, D) Immunofluorescence analysis of cell line 427/p2T7ARL6 grown in the presence of tetracycline for 24 h, probed with anti-PFR1/2 (C) or anti-α-tubulin (D) (shown in green) and co-stained with DAPI (blue). Bar, 5 μm. The bottom right panel in (C) is an enlarged view (× 2.5) of the region marked by a grey box in the bottom left panel. (E, F) Paraflagellar rod length (E) and cell body length (F) in BSF parental line Lister 427 (wt) and cell line 427/p2T7ARL6 (RNAi) grown in the presence of tetracycline for 0–72 h. Mean values are shown as horizontal lines (± SD) (n = 100). Statistical analyses were performed using 1 way ANOVA. Asterisks represent statistical significance compared to the wild-type (wt) sample (** = p < 0.01, *** = p < 0.001). All data shown are representative of experiments using at least 3 independent RNAi clones.