Additional file 4: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Differentially expressed genes (≥ 2-fold) in successive life-cycle stages. Sl: slender bloodstream forms; St: stumpy bloodstream forms; Ea: early procyclic forms; La: late procyclic forms. (XLSX 5584 kb

Additional file 4: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Differentially expressed genes (≥ 2-fold) in successive life-cycle stages. Sl: slender bloodstream forms; St: stumpy bloodstream forms; Ea: early procyclic forms; La: late procyclic forms. (XLSX 5584 kb

Additional file 6: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Differential expression ≥2-fold in early and late procyclic forms based on 3′ untranslated regions. Ea: early procyclic forms; La: late procyclic forms. (XLSX 44 kb

Additional file 5: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Stage-specific transcripts enriched ≥2-fold compared to all other stages. Sl: slender bloodstream forms; St: stumpy bloodstream forms; Ea: early procyclic forms; La: late procyclic forms. FC: fold change. (XLSX 59 kb

Additional file 2: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Mean expression values (reads per million) for all transcripts. Sl: slender bloodstream forms; St: stumpy bloodstream forms; Ea: early procyclic forms; La: late procyclic forms. (XLSX 762 kb

Additional file 1: of RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

Summary of mapping information. (DOCX 45 kb

RNAi against TAC102 in γagainsBSF cells affects kDNA segregation but not cell growth.

<p><b>A</b>–growth curve of cells uninduced (–Tet) and induced (+Tet) for TAC102 RNAi. Inset: northern blot probed for TAC102 and 18S rRNA (loading control). RNA was isolated from cells that were uninduced (−) or induced for two days (+). <b>B</b>–epifluorescence images (DAPI staining) of cells upon induction of RNAi against TAC102 for two days. Scale bar 5 n o <b>C</b>–percentage of cells with different k-n-combinations within the course of TAC102 RNAi.</p

RNAi against TAC102 in BSF cells causes missegregation and loss of kDNA.

<p><b>A</b>–growth curve of cells uninduced (−Tet) and induced (+Tet) for TAC102 RNAi. Inset: northern blot probed for TAC102 and 18S rRNA (loading control). RNA was isolated from cells that were uninduced (−) or induced for two days (+). The western blot under the growth curve demonstrates downregulation of TAC102 protein upon induction of RNAi. α Tubulin was used as a loading control. <b>B</b>–immunofluorescence images showing missegregation and loss of kDNA as well as the unchanged mitochondrial morphology upon induction of RNAi against TAC102. DNA is stained with DAPI (cyan). Mitochondria are visualized by staining for the mitochondrial heat-shock protein 70 (mtHSP70, red). Scale bar 5 he <b>C</b>–percentage of cells with different k-n-combinations within the course of TAC102 RNAi. <b>D</b>–intensity of the kDNA signal measured by DAPI staining. <b>E</b>–the relative amount of minicircle DNA decreases within the course of TAC102 RNAi, according to Southern blotting. Tubulin was used for normalization and the amount of minicircle DNA in non-induced cells (day 0) was taken as 100%. A representative Southern blot is shown on the right side of the graph.</p

Ultrastructure of kDNA upon RNAi against TAC102 in BSF cells revealed by transmission electron microscopy.

<p>bb, basal body; mm, mitochondrial membrane; edm, electron dense material; −Tet, non-induced cells; +Tet, cells with TAC102 RNAi induced for two days. <b>A,B</b>–kDNA in a non-induced cells. <b>C,D</b>–examples of enlarged kDNA in cells induced for TAC102 RNAi for two days. The arrows point at additional patches of kDNA. <b>E</b>–example of a cell induced for TAC102 RNAi for two days that has lost the kDNA, instead only a small patch of electron dense material (edm) can be seen surrounded by the mitochondrial membrane (mm) and localized in the proximity to the basal body (bb). <b>F</b>–an example of a cell induced for TAC102 RNAi for two days that has lost the kDNA and does not have any detectable electron dense material (edm); however, the mitochondrial membrane (mm) and the basal body (bb) are seen well. <b>G</b>–quantification of the kDNA diameter, measured as shown in <b>A</b> (white bar), in non-induced and induced cells.</p

The myc:full version of TAC102 is able to partially compensate for the loss of the endogenous TAC102 but the myc:ΔN is not.

<p>The full-length (myc:full) or the N-terminally truncated (myc:yc: version of TAC102 was expressed with a triple myc-tag at the N-terminus in PCF cells that contain a construct for inducible RNAi against the 3’-UTR of the endogenous TAC102. Immunofluorescence images show the localization of the tagged proteins. DNA is stained with DAPI (cyan) and myc-tagged proteins (visualized by α-myc antibody) are shown in magenta. Arrows show additional foci of TAC102 accumulation. Stars show ancillary kDNAs. Scale bar 5 addiFor each of the two cell lines, growth curves of the cells upon expression of the tagged proteins/knockdown of the endogenous TAC102 are shown on the right side of the immunofluorescence images. Under the growth curves (left side) are western blots that show expression levels of the tagged versions of TAC102 in comparison to those of the endogenous protein (endo). EF1o)stern d as a loading control. Under the growth curves (right side) are western blots of digitonin fractionations. ATOM (a mitochondrial protein) and EF1α (a cytosolic protein) are used as fractionation controls. T, total cell lysate; S, supernatant; P, pellet. Fractionations were performed on day 5 of induction.</p