FIG 1.

LC8 silencing has pleiotropic effects on BF trypanosomes. (A) Growth curve analysis of a representative clonal smLC8 BF cell line in the absence (−dox) and presence (+dox) of the LC8 knockdown-inducing compound doxycycline. (B) Total RNA prepared from noninduced (ni) and 1-day-induced cells was reverse transcribed with oligo(dT) and analyzed by semiquantitative PCR using LC8-, α-tubulin-, and VSG2-specific oligonucleotides. rRNA was detected by ethidium bromide staining of total RNA. (C) Whole-cell lysates of noninduced and 1- or 2-day-induced cells were analyzed by immunoblotting using LC8- or CITFA6-specific antibodies or, as a loading control, TFIIB-specific antibodies. (D) Flow cytometry analysis, without gating, of noninduced (blue) and 1-day-induced cultures (red). Propidium iodide staining intensity (x axis), which measures DNA content per cell, and cell counts (y axis) from one representative experiment are shown. K, thousands. (E) Indirect fluorescence microscopy of noninduced and 1-day-induced cells. Representative single cells from each culture were imaged using both phase-contrast and DAPI fluorescence. Small and large areas of DAPI intensity represent kinetoplasts and nuclei, respectively. Scale bars, 10 μm.