Figure S1 from Prostaglandin F2α synthase in <i>Trypanosoma cruzi</i> plays critical roles in oxidative stress and susceptibility to benznidazole

<b>OYE protein expression in Bz-sensitive (61Scl1) and Bz-resistant (61Rcl4).</b> Western blot using specific anti-OYE in parasites sensitive and resistant to Bz. GAPDH and Cruzipain proteins were used as load controls. Quantification was performed with GAPDH as the normalizer

Viability of TcNTH1 transfected epimastigotes submitted to oxidative stress.

<p>TcNTH1 overexpressing epimastigotes and its control (parasites transfected with empty vector) were treated for 30 min with different H₂O₂ initial concentrations (A) or with a glucose-glucose oxidase system producing sustained H₂O₂ concentrations for 24 hours (B). Viability was determined by AlamarBlue assays. *p value: 0,01</p

Number of differentially expressed genes in definite time points after <i>T. cruzi</i> irradiation.

<p>A) Scatter chart showing the distribution of fold-change values for statistically significant up-regulated genes over time points. B) Number of genes differentially expressed over time. ○: up-regulated, □: down-regulated. i.a.i: immediately after irradiation.</p

<i>T. cruzi</i> nuclear genes expression profiles.

<p>A) Heatmap of nuclear genes differentially expressed after <i>T. cruzi</i> irradiation. Shades of green indicate down-regulated and shades of red up-regulated genes. B) Clusters of genes based on similar expression profiles over time. Red lines: average fold-change values.</p

Expression of TcNTH1 DNA glycosylase in <i>T</i>. <i>cruzi</i> cellular forms.

<p><b>A:</b> TcNTH1 polyclonal antibodies prepared in mice specifically identifies a purified recombinant TcNTH1 (lane 1) and a TcNTH1 expressed in recombinant bacterial homogenates (lane 2). This protein was not recognized in non-expressing bacteria (lane 3). <b>B:</b> Western blot detection of TcNTH1 in total protein homogenates from epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3). Lanes 4 and 5 correspond to TcNTH1 purified from transformed <i>E</i>. <i>coli</i> and to the same protein from recombinant over-expressing <i>T</i>. <i>cruzi</i> epimastigote homogenate, respectively. <b>C:</b> Same as B but using an anti-HIS antibody. <b>D:</b> Loading control for epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3) using an alpha-tubulin antibody. All electrophoretic separations were performed in 12%SDS-PAGE.</p

Time course expression of kDNA genes after <i>T. cruzi</i> cells irradiation.

<p>COI: Citochrome oxidase I; ND-1: NADH-dehydrogenase 1; ND-5: NADH-dehydrogenase 5; MURF-1 = Mitochondrial undefined reading frame 1. i.a.i: immediately after irradiation.</p

Fold-change of differentially down-regulated expressed genes coding for proteins with known function.

<p>Bolded fold-changes: statistically significant values. i.a.i.: immediately after irradiation.</p

Categories of differentially expressed genes and their distributions along the time after <i>T. cruzi</i> irradiation.

<p>White: genes coding for hypothetical proteins. Light gray: obsolete sequences. Dark gray: genes coding for proteins with known function. Black: RHS genes. i.a.i: immediately after irradiation.</p

Frequency of RHS genes and non-RHS genes up-regulated in response to treatment with gamma rays over time.

<p>White squares: frequency of up-regulated RHS genes. Black circles: frequency of up-regulated genes, excluding RHS genes. i.a.i: immediately after irradiation. **** p<0.0001.</p

The catalytic residues disposition and its effects on lesion recognition by TcNTH1.

<p><b>A:</b> Protein-DNA complexes of <i>G</i>. <i>stearothermophilus</i> Endonuclease III (PDB 1P59, cyan) as determined by X-ray crystallography and TcNTH1 from <i>T</i>. <i>cruzi</i> (magenta) as determined by molecular docking. Only protein backbones are shown. <b>B:</b> The same protein-DNA complexes in a different view and depicting the protein surface. Structurally divergent residues are labeled to suggest regions of interest for further analyses. The DNA-interacting region is circulated and the lesion site is shown inside the rectangle. The DNA molecule represented is from 1P59, which is in a similar position when compared to the TcNTH1-DNA complex, as shown in A.</p