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. 2020 Jun 10;18(6):e3000741. doi: 10.1371/journal.pbio.3000741

Fig 10. Elevated ROS generated during the differentiation of T. brucei are crucial for driving forward RBP6-induced differentiation.

Fig 10

(A) Metabolites glutathione and glutathione disulfide as detected by LC-MS analyses. The size of the bars represents the total abundance of the metabolite (mean ± SD, n = 4, ***P < 0.001). (B) Mitochondrial and cellular ROS detection reagents (MitoSox and H2DCFDA, respectively) were quantified by FACS (means ± SD, n = 5, ***P < 0.001, ****P < 0.0001). (C) Representative growth curves of RBP6OE and RBP6OE_catalase cells induced by tetracycline. Total number of experiments n = 5. The inset shows subcellular localization of v5-tagged catalase in RBP6OE_catalase cells induced for 48 hours. Immunoblots were labeled with anti-v5, anti-adenosine phosphoribosyl transferase (APRT), and anti-mt hsp70 antibodies to visualize catalase, cytosolic APRT, and mitochondrial localized hsp70, respectively. (D) Time line for the appearance of epimastigotes and metacyclic cells upon induction of RBP6OE and RBP6OE_catalase. Total number of experiments n = 3. (E) Western blot analysis of whole-cell lysates from RBP6OE and RBP6OE_catalase cells using available antibodies. (F) FACS analyses of RBP6OE and RBP6OE_catalase cells treated with 5 mM bathophenanthroline disulphonic acid (BPS) and labeled with polyclonal anti-BARP and anti-procyclin antibodies. (G) The Δψm of RBP6OE_catalase cells post induction measured by flow cytometry using TMRE (means ± SD, n = 4), *P < 0.05. (H) Mitochondrial ROS detection reagent MitoSox in RBP6OE_catalase cells quantified by FACS (means ± SD, n = 5), *P < 0.05, ***P < 0.001. (I) Cellular ROS detection reagent H2DCFDA in RBP6OE_catalase cells quantified by FACS (means ± SD, n = 5). Underlying data plotted in panels A, B, C, D, F, G, H, and I are provided in S1 Data. AOX, alternative oxidase; APRT, adenine phosphoribosyl transferase; BARP, brucei alanine-rich protein; CYT, cytosol; hsp70, heat shock protein 70; H2DCFDA, 2′,7′-dichlorofluorescin diacetate; LC-MS, liquid chromatography–mass spectrometry; ns, statistically not significant; ORG, organellar fraction; RBP6, RNA binding protein 6; ROS, reactive oxygen species; SCoAS, succinyl Co-A synthetase; TMRE, tetramethyl rhodamine ethyl ester; WCL, whole-cell lysate.