Figure 2.

TbPPL2 Is Essential and Cell-Cycle Regulated

(A–D) Western blot analysis is shown (A and C) with anti-cMyc and cell lysates prepared from representative TbPPL1^Myc and TbPPL2^Myc RNAi cell lines, respectively, following RNAi induction with tetracycline (1 μg/ml) for the times indicated. Equivalent Coomassie-stained gels serve as loading controls. Growth curves are shown (B and D) of TbPPL1^Myc and TbPPL2^Myc RNAi cell lines, respectively, grown in the presence or absence of tetracycline (1 μg/ml). Cultures were diluted to 10⁵ cells/ml every 24 hr as appropriate. Representative growth curves are shown for four experiments performed in duplicate, each with independent cell lines (three without native-tagging). Error bars represent one standard deviation.

(E) Immunofluorescent detection of TbPPL2^Myc with anti-cMyc. DNA was counterstained with DAPI. Nucleus (n), kinetoplasts (k). Scale bar is 5 μm.

(F) Cells with TbPPL2^Myc detectable by immunofluorescence were scored for cell-cycle position. G₁/S, 1 nucleus and 1 kinetoplast (1n1k); G₂, 1 nucleus and 2 kinetoplasts (1n2k); post-M, 2 nuclei and 2 kinetoplasts (2n2k). n = 200; error bars, standard deviation.