JCS016741 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. (A) Amino acid sequence alignment of SPT1 proteins of Trypanosoma brucei (TbSPT1, GeneDB number Tb927.4.1020), Trypanosoma cruzi (TcSPT1, accession number Tc001047053508257.10, Tc001047053503929.79, Tc001047053506405.50), Leishmania major (LmSPT1, accession number LmjF34.3740), Homo sapiens (HsLCB1, PubMed accession number O152069), and Saccharomyces cerevisiae (ScLCB1, PubMed accession number P25045). The VectorNTI alignment algorithm was used. Amino acids highlighted in yellow and blue are identical in all or most sequences, respectively; green highlighting represents conservative substitutions. (B) Amino acid sequence alignment of SPT2 proteins of Trypanosoma brucei (TbSPT2, GeneDB accession number Tb10.70.3220), Trypanosoma cruzi (TcSPT2, GeneDB accession number accession number Tc00.1047053503453.100), Leishmania major (LmSPT2, GeneDB accession number LmjF35.0320), Homo sapiens (HsLCB2, PubMed accession number 15270), and Saccharomyces cerevisiae (ScLCB2, PubMed accession number P40970). The VectorNTI alignment algorithm was used. Amino acids highlighted in yellow and blue are identical in all or most sequences, respectively; green highlighting represents conservative substitutions. The putative transmembrane domain of TbSPT2 is indicated by a red underline, based on homology with yeast LCB2. A red asterisk marks the putative PLP-binding lysine residue within the catalytic site motif 381 GTFTKSFG 389 (red box) in the TbSPT2 sequence (based on sequence for yeast (Hanada, 2003), and L. major (Zhang et al., 2003)). The region targeted in RNAi experiments is underlined in blue.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Identification of the phospholipid ion species by ESI-MS/MS. Lipids extracted by the modified Folch’s method were analyzed by negative-ion mode ESI-MS/MS. (A-C) Assigned fragments for (A) IPC, (B) diacyl-PI, and PC and (C) alkyl-acyl-PI species are indicated. Gro_cP, glycerol-cyclic phosphate; mI_cP, myo-inositol-cyclic phosphate; mIP, myo-inositol-phosphate; C16:0, palmitic acid; C18:2, linoleic acid; C18:1, oleic acid; C18:0, stearic acid; OH-C18:1, mono-oxidized oleic acid; M, molecular mass; Cl⁻, chlorine; m/z, mass to charge ratio.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. GC-MS analysis of sterols in TbSPT2 RNAi cell line and myriocin (Myr)-treated 29-13 T. brucei. Sterols extracted by the modified Folch’s method and purified using a column with silica gel resin were analyzed by GC-MS, using sitosterol (Sit) as an internal standard, and cholesterol (Cho), lanosterol (Lan), and ergosterol (Erg) as external standards, to determine the type and amount of sterols present in the cells. The major T. brucei sterols detected were Cho, ChoE, and Lan but very little Erg. Myriocin-treated 29-13 cells and both tetracycline-induced and uninduced TbSPT2 RNAi lines contain elevated cholesterol levels relative to control 29-13 cell line.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Percentage of cells with different amounts of nuclei and kinetoplasts. Cells were assessed for DNA and kinetoplast content at days 0-3 after induction of TbSPT2 RNAi with 1 µg/ml tetracycline. TbSPT2 RNAi cells were fixed with paraformaldehyde for 30 minutes and stained with DAPI to visualize nuclear content. The experiment was repeated three times, with at least 200 cells counted per experiment. A representative experiment is shown.

• Movie 1 -

  Movie 1. (Left) unininduced TbSPT2 cells have normal morphology and motility. (Right) The majority of Tet-induced TbSPT2 RNAi cells are grossly abnormal after 6 days of treatment. The various phenotypes include multi-flagellated cells fused at the posterior end (one example enters the screen at 12 o’clock at 11 seconds, another enters the screen at 10 o’clock at 8 seconds), spherical syncytia possessing multiple peripheral flagella that lack directional movement (one example visible at the center of the screen, another visible at 11 o’clock at 15 seconds), cells with enlarged vacuoles that retain directional movement (e.g. cell visible at 6 o’clock at the start of the movie, as well one visible at 9 o’clock at 12 seconds), extremely long cells with a branched extended posterior resembling cells depleted of CRK1 and CRK2 (Tu and Wang, 2005) (visible at 12 o’clock at 8 seconds), and debris from dying cells.