Figure 4.

Trypanosoma cruzi-induced ROS is a potent inducer of SASP in NIH-3T3 fibroblasts. NIH-3T3 fibroblasts were infected overnight with culture-derived T. cruzi trypomastigotes (MOI 5:1). After washing step to eliminate extracellular parasites, cultures were treated with (A) 50% supernatants from non-infected or (B) T. cruzi-infected cultures (conditioned medium) plus 50% of fresh medium. After 3 days, the number of T. cruzi-infected or non-infected fibroblasts were quantified by optical microscopy. Dead cells were evaluated by Trypan blue exclusion assay. (C–E) NIH-3T3 fibroblasts were loaded with the probe DCFH-DA, washed and infected with T. cruzi concomitantly with addition of antioxidants NAC (20 mM) and DFO (40 µM) during infection period (MOI 5:1). After overnight infection, cultures were washed to eliminate extracellular parasites and cultured for additional 3 days. (C) Assessment of ROS accumulation after T. cruzi infection in NIH-3T3 fibroblasts by fluorescence. Results indicate arbitrary units of fluorescence. (D) Evaluation of IL-6 in culture supernatant by ELISA. (E) Evaluation of soluble SA-β-gal activity at pH 6.0 in cell lysates. (F) Assessment of extracellular parasitic load (trypomastigote forms) in culture supernatants over 10 days. Data are presented as the mean ± SE of at least six biological replicates and analyzed by unpaired, two-tailed Student’s t-test (C,D), and non-parametric Mann–Whitney test (E). For (A,B,F), data were log-transformed and analyzed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; NS, statistically not significant, p > 0.05. Data are representative of at least three independent experiments with similar results. Abbreviations: MOI, multiplicity of infection; Ctrl, control; ROS, reactive oxygen species; NAC, N-acetylcysteine; DFO, deferoxamine; DCFH-DA, dichloro-dihydro-fluorescein diacetate; h, hours; ROS, reactive oxygen species; SASP, senescence-associated secretory phenotype; SA-β-gal, senescence-associated β-galactosidase.