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 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

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 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 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

Differential expression of markers in early and late procyclic culture forms.

<p>(A) Northern blot analysis of adenylate cyclases Tb927.5.330 (AC 330) and Tb.927.5.320/285b (AC 320). GPEET was used to verify that the cultures correspond to early and late procyclic forms, respectively. Signals were quantified using a PhosphoImager and normalised against 18S rRNA as described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004493#ppat.1004493-Flck1" target="_blank">[47]</a>. Signals in early procyclic forms were set at 1. (B) Immunofluorescence analysis reveals that GPEET and calflagin are co-expressed by early procyclic culture forms and are not detectable in late procyclic culture forms. Scale bar: 10 µm. (C) Quantitative RT-PCR performed using RNA from early procyclic culture forms and late procyclic culture forms 11 days after removal of glycerol from the medium <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004493#ppat.1004493-Vassella2" target="_blank">[4]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004493#ppat.1004493-Knsel1" target="_blank">[7]</a>. RQ: Relative quantification. Expression levels in early procyclic forms are set at 1. α-tubulin was used to normalise mRNA levels. Error bars are ΔCt standard errors. AC 330: Tb927.5.330 3′ UTR; AC 320: Tb927.5.320/285b 3′ UTR; HK1: 3′ UTR of Tb927.10.2010: HK2: 3′ UTR of Tb927.10.2020; Calflagin: coding region of Tb927.8.5460, Tb927.8.5440, Tb927.8.5465, Tb927.8.5470. PPT: coding region of Tb927.1.2850, Tb927.1.2880 (putative pteridine transporters).</p

GPEET is not essential for SoMo.

<p>(A) 2×10⁵ ΔGPEET cells, cultured with or without glycerol, were inoculated onto a plate containing 20 mM glycerol. Four days post plating only the cells cultured in medium containing glycerol showed SoMo. The scale bar is 1 cm. (B) 4×10⁵ ΔGPEET/GFP cells, cultured in medium containing glycerol, were inoculated onto a plate containing 20 mM glycerol. Four days post plating a community lift was stained with α-GFP antibody.</p

Differential expression of early and late procyclic form markers in tsetse flies.

<p>A. GPEET and calflagin are co-expressed by early procyclic forms 3 days post infection (DPI), but neither is detectable in late procyclic forms 12 DPI. Trypanosomes were isolated from tsetse fly midguts, fixed with formaldehyde and glutaraldehyde and permeabilised with Triton-×100. Immunofluorescence was performed with anti-GPEET and anti-calflagin antisera. Scale bar: 10 µm. B. Quantitative RT-PCR was performed with RNA isolated from infected tsetse flies 3 and 12 days post infection. Gene designations are the same as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004493#ppat-1004493-g006" target="_blank">Figure 6</a>. RQ: relative quantification. α-tubulin was used to normalise mRNA levels. Error bars are ΔCt standard errors.</p

Deletion mutants with defects in salivary gland infection rates are SoMo-positive.

<p>A and B. MKK1 and PSSA-2 deletion mutants were cultured with or without glycerol. 4×10⁵ cells from each culture were inoculated onto plates containing 20 mM glycerol. The scale bar is 1 cm. Four days post plating community lifts were incubated with α-EP and α-GPEET antibodies. C. 2×10⁵ cells of the procyclin null mutant (Δproc), obtained by differentiation of bloodstream forms, were inoculated onto a 0.4% agarose plate containing 20 mM glycerol. A photograph was taken 4 days post plating.</p