FIG 5.

Effect of inhibition of TbPI-PLC1 expression by tetracycline-inducible RNAi on cell growth. (A) Growth of PCF trypanosomes in the absence (squares) or presence (circles) of 1 μg/ml tetracycline for the indicated numbers of days. Values are means of triplicate determinations for a representative experiment. (B) Northern blot analysis of TbPI-PLC1 RNAi of PCF trypanosomes grown in the absence (−) or presence (+) of tetracycline (Tet). (Top) Total RNA was subjected to gel electrophoresis before transfer to a nylon membrane and then hybridized with a ³²P-labeled probe corresponding to the TbPI-PLC1 coding sequence. (Bottom) Tubulin is shown as a loading control. (C) PIP₂ hydrolysis was measured in PCF trypanosome lysates as described in Materials and Methods, using 5 μM PIP₂ and different Ca²⁺ concentrations. (D) Activity in lysates from the parent line (WT), trypanosomes overexpressing TbPI-PLC1 (OE), or RNAi transgenic trypanosomes (RNAi) in the absence (−) or presence (+) of tetracycline (Tet). Data are means and SEM (n = 3). ***, P < 0.05 compared to wild-type lysates (Student's t test).