The mutant cells were either kept in Trolox-supplemented medium or transferred into medium containing 100 µM Dfx, 100 nM ferrostatin-1 or 200 nM liproxstatin-1 or no addition. (A) Every hour viable …
The cells were incubated in the presence of (A) different concentrations of Trolox, (B) various concentrations of Dfx, (C) in medium supplemented with 10 µM Trolox or 20 µM Dfx alone or in …
The parasites were incubated for 1 or 2 hr in medium ± Trolox, treated with MitoTracker (red) and subjected to immunofluorescence microscopy using antibodies against (A) mtTXNPx (green) or (B) VDAC …
The Px I-III KO cells were incubated ± Trolox for up to 4 hr, stained with MitoTracker or propidium iodide and subjected to flow cytometry. (A) Forward- (FSC) and side-scatter (SSC) DotPlots of …
Parasites kept in the presence or absence of Trolox were fixed, processed and subjected to transmission electron microscopy as described under Materials and methods. Electron micrographs of …
Electron micrographs of Px I-III KO parasites kept for (A) 1 hr and (B) 2 hr in Trolox-free medium. (A) The intact elongated mitochondrion is darker than the cytosol. Other subcellular structures …
(A) The cells were pre-loaded with MitoSOX in Trolox-containing medium, transferred into medium ± Trolox, incubated for 2 hr, and stained with MitoTracker Green. Nuclear (large dot) and kinetoplast …
The Px I-III KO cells were loaded with MitoSOX for 10 min in Trolox-supplemented medium, washed with PBS and re-suspended in medium ± Trolox. After 5 hr incubation, the cells were stained with DAPI …
The cells were incubated ± Trolox in (A) SDM-79 medium or (B) MEM-Pros medium. After 0 to 4 hr, aliquots of each sample were removed and (A) cells with normal morphology or only highly motile cells …
(A) Px I-III KO parasites in Trolox-supplemented medium were incubated for 1.5 hr with MitoPerOx, stained with MitoTracker Green and subjected to life cell fluorescence microscopy. Representative …
(A) Total lysates of 5 × 106 Px I-III KO/SODA-myc cells cultured for 18 hr ± tetracycline (Tet) were subjected to Western blot analysis using antibodies against myc as well as aldolase for loading …
(A) Cells were pre-loaded with 150 nM RPA or 10 nM RPAC in PBS, re-transferred into Trolox-supplemented medium, stained with MitoTracker Green and subjected to life cell imaging. Merge, overlay of …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Trypanosoma brucei brucei) | sodA | TriTrypDatabase ID: Tb427.05.3350 | ||
Cell line (Trypanosoma brucei brucei) | WT PC and BS | PMID: 9108552 | WT PC and BS 449 Lister strain 427 | Culture-adapted T. brucei strains stably expressing the tetracycline repressor |
Cell line (Trypanosoma brucei brucei) | PC Px I-III KO | PMID: 26374473 | PC WT cells in which both alleles of the complete px locus are replaced by resistance casettes (blasticidin and puromycin) | |
Cell line (Trypanosoma brucei brucei) | PC Px I-III KO/SODA-myc | this work | PC Px I-III KO cells that contain a Tet-inducible construct for SODA-c-myc2 overexpression | |
Antibody | Rabbit anti-cTXNPx | PMID: 20826821 | IF (1:1000) | |
Antibody | Guinea pig anti-mTXNPx | PMID: 29413965 | IF (1:1000) | |
Antibody | Rabbit anti-aldolase | Christine Clayton, Heidelberg, Germany | WB (1:20000) | |
Antibody | Rabbit anti-VDAC | André Schneider, Bern, Switzerland | IF (1:500) | |
Antibody | Mouse anti-c-Myc (monoclonal) | Santa Cruz Biotechnology | sc-40, RRID:AB_627268 | WB, IF (1:200) |
Antibody | HRP-conjugated goat anti-mouse IgGs | Santa Cruz Biotechnology | sc-2005, RRID:AB_631736 | WB (1:5000) |
Antibody | HRP-conjugated goat anti-rabbit IgGs | Santa Cruz Biotechnology | sc-2004, RRID:AB_631746 | WB (1:10000) |
Antibody | Alexa Fluor 488 goat anti-guinea pig IgGs | Thermo Fisher Scientific | A11073, RRID:AB_142018 | IF (1:1000) |
Antibody | Alexa Fluor 488 goat anti-mouse IgGs | Thermo Fisher Scientific | A11001, RRID:AB_2534069 | IF (1:250) |
Antibody | Alexa Fluor 488 goat anti-rabbit IgGs | Thermo Fisher Scientific | A11008, RRID:AB_143165 | IF (1:1000) |
Antibody | Alexa Fluor 594 goat anti-rabbit IgGs | Thermo Fisher Scientific | A11012, RRID:AB_141359 | IF (1:1000) |
Recombinant DNA reagent | pHD1700/grx2- c-myc2 (plasmid) | PMID: 20826822 | ||
Recombinant DNA reagent | pHD1700/sodA- c-myc2 (plasmid) | this work | ||
Sequence-based reagent | 5'CGATAAGCTTATG AGGTCTGTCATGATGC3' | this work | Primer for amplification of sodA from genomic T. brucei DNA | |
Sequence-based reagent | 5'CGATGGATCCCTT CATAGCCTGTTCATAC3' | this work | Primer for amplification of sodA from genomic T. brucei DNA | |
Peptide, recombinant protein | T. brucei trypanothione reductase (TR) | PMID: 24788386 | ||
Peptide, recombinant protein | T. brucei tryparedoxin (Tpx) | PMID: 22275351 | ||
Peptide, recombinant protein | T. brucei peroxidase (Px) | PMID: 18684708 | ||
Chemical compound, drug | trypanothione and trypanothione disulfide | PMID:19477177 | ||
Chemical compound, drug | Chlorhexidine diacetate Monohydrate | Fluka | 24800 | |
Chemical compound, drug | (1S,3R)-RSL3 | José Pedro Friedmann Angeli and Marcus Conrad, Munich, Germany and Cayman Chemical | CAS: 1219810-16-8 | |
Chemical compound, drug | Racemic mixture of RSL3 | José Pedro Friedmann Angeli and Marcus Conrad, Munich, Germany | ||
Chemical compound, drug | Ferrostatin-1 | Sigma-Aldrich | SML0583 | |
Chemical compound, drug | Liproxstatin-1 | Sigma-Aldrich | SML1414 | |
Chemical compound, drug | Trolox | Sigma-Aldrich | 238813 | |
Chemical compound, drug | Iron(III) chloride hexahydrate, FeCl3 × 6H2O | Merk | 31232 | |
Chemical compound, drug | Deferoxamine mesylate | Sigma-Aldrich | D-9533 | |
Chemical compound, drug | 8-Hydroxyquinoline | Sigma-Aldrich | 252565 | |
Chemical compound, drug | (±)-α-Tocopherol | Sigma-Aldrich | T3251 | |
Chemical compound, drug | ATPlite 1step solution | Perkin Elmer | 6016731 | |
Chemical compound, drug | Hoechst 33342 | Walter Nickel, Heidelberg, Germany | ||
Chemical compound, drug | 4’,6-Diamidino-2 -phenylindole (DAPI) | Sigma-Aldrich | D-8417 | |
Chemical compound, drug | Rhodamine B-[(1,10-phenanthroline-5-yl)- aminocarbonyl]benzyl ester (RPA) | Squarix Biotechnology | ME043.1 | |
Chemical compound, drug | Rhodamine B-[(phenanthren-9-yl) -aminocarbonyl]- benzylester (RPAC) | Squarix Biotechnology | ME046.1 | |
Chemical compound, drug | BODIPY 581/591 C11 | Molecular Probes | D3861 | |
Chemical compound, drug | MitoPerOx | Mike Murphy, Cambridge, UK | ||
Chemical compound, drug | MitoTracker Red CMXRos | Molecular Probes | M7512 | |
Chemical compound, drug | MitoTracker Green FM | Molecular Probes | M7514 | |
Chemical compound, drug | MitoSOX Red | Molecular Probes | M36008 | |
Chemical compound, drug | Propidium iodide (PI) | Molecular Probes | P3566 | |
Chemical compound, drug | 2',7'-Dichlorodihydro fluorescein-diacetate (H2DCFDA) | Molecular Probes | D399 |
Trypanocidal activity and in vitro inhibitory potency of RSL3.
(A) Bloodstream T. brucei were cultured for 1, 2 or 3 days in the presence of 100 µM Trolox, 200 nM Liproxstatin-1 or 100 nM Ferrostatin-1 as well as different RSL3 concentrations and then subjected to plate reader-based ATPlite measurements. Chlorhexidine, a trypanocidal compound and known inhibitor of trypanothione reductase (TR) (Meiering et al., 2005; Beig et al., 2015), served as positive control. 1The data are the mean of at least two independent series of experiments each conducted in triplicate with standard deviations (SD). 2The values are the mean of an experiment conducted in triplicate with SD. (B) NADPH, T(SH)2, TR, and Tpx ± Px were incubated with 40 µM RSL3. After different times, the assays were started by adding (a) 100 µM H2O2 or (b) Px and H2O2. The data were derived from at least double determinations which varied by ≤ 10%.