Figure 4. RNAi against all PAD genes reduces differentiation.
- (a) PAD-array expression in parental or PAD-RNAi lines (±doxycycline, D+, D) after 16h at 20°C. Tubulin, loading control.
- (b) EP-procyclin in Parental (‘P’) and PAD-RNAi stumpy-forms grown ±doxycycline (D+, D−) after 16h at 20°C, then incubated at 27°C with a CA titration. Means (±s.e.m.) of three experiments are shown, as is the % reduction in EP-procyclin in the PAD-RNAi cells compared with parental cells.
- (c) Flow-cytometry of EP-procyclin expression in PAD-RNAi or parental cells incubated with 0.1mM or 1mM CA. Cold-induced hypersensitivity to CA is ablated in the PAD-RNAi line; at ≥1mM CA both populations differentiate, although this is reduced in the RNAi line. Full flow cytometry data are available in Supplementary Fig. 7.