Fig 2: T. cruzi infection induces inflammation and cell death in adipose tissue via necrosis and apoptosis.A. Immunoblot analysis of (A) markers of inflammation (infiltrated macrophages (F4/80), and cytokines (INF? and TNFa)), and (B) cell death (BNIP3 (a marker of necrosis), and cleaved Caspase3 (a marker of apoptosis)) in the adipose tissue of indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab -) n = 8). The change in protein levels were normalized to the levels of Guanosine nucleotide dissociation inhibitor (GDI) and plotted column scatter graph. The error bars represent SEM. A.U. indicates arbitrary unit. *p=0.05, **p=0.01 or ***p=0.001 compared with uninfected fat-unablated. #p=0.05, ##p=0.01 or ###p=0.001 compared with infected fat-unablated.

Fig 3: BNIP3 protects mitochondrial homeostasis in mouse ESCs.A Real-time PCR analysis of mtDNA in WT and mitophagy receptor knockout ESCs. Data are shown as mean ± SD, n = 3; *P < 0.05; Student’s t test. B Analysis of mitochondrial mass (Mito-mass) in WT and mitophagy receptor knockout ESCs. MitoTracker Green was used for Mito-mass detection. Data are shown as mean ± SD, n = 3; *P < 0.05; Student’s t test. C Mitochondrial membrane potential of WT and mitophagy receptor knockout ESCs. Data are shown as mean ± SD, n = 3; **P < 0.01; Student’s t test. D ROS generation in WT and mitophagy receptor knockout ESCs. Data are shown as mean ± SD, n = 3; **P < 0.01; Student’s t test. E ATP generation in WT and mitophagy receptor knockout ESCs. Data are shown as mean ± SD, n = 3; **P < 0.01; Student’s t test. F Oxygen consumption rates (OCRs) of WT and Bnip3-/- ESCs treated sequentially with oligomycin, FCCP, and rotenone.

Fig 4: Recovery of HIF-1a levels in BNIP3-silenced cells restores melanoma glycolytic phenotype and growth in vitro (related to Fig 7) A, BMurine (A) and human (B) HIF1? transcript levels measured by qPCR in B16-F10 cell lysates collected 48 h after plating (n = 5). They were analyzed against shCntl Luc (A) or shCntl HIF-1a-AA (B) using a one-sample t-test except human HIF1A in shBNIP3 Luc that was analyzed with Wilcoxon rank test.CImmunoblot detection of PHD2 and ACTIN levels from B16-F10 cell lysates collected 48 h after plating (n = 4). Densitometric quantifications are shown below each corresponding band they were analyzed using a one-sample t-test against shCntl Luc.DBasal respiration rates of B16-F10 cells assayed in galactose (25 mM galactose, 2 mM glutamine) medium using the Seahorse technology (n = 4).EClonogenic growth of B16-F10 cells 10 days after seeding. Data information: All quantitative data are mean ± SEM. Transcript expression is represented as the fold change relative to their corresponding shCntl value. *P < 0.05 **P < 0.01 when compared against shCntl Luc and $$$ P < 0.001, $$$$P < 0.0001 when compared against shCntl HIF-1a-AA.

Fig 5: BNIP3 depletion impairs mitochondrial clearance and rewires the glycolytic metabolism of melanoma cells AConfocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n = 27 cells) or deficient (shBNIP3, n = 31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Co-localization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann–Whitney’s non-parametric test. Scale bars represent 10 µm.BSchematic representation of the metabolic approaches used to characterize B16-F10 cells 1H-NMR stands for proton-based nuclear magnetic resonance, GC-MS for gas chromatography coupled to mass spectrometry, OCR for oxygen consumption rate and ECAR for extracellular acidification rate.CBasal OCR per µg of protein of B16-F10 cells in the presence of galactose (25 mM galactose, 2 mM glutamine) medium using the Seahorse technology (n = 3) analyzed using an RM one-way ANOVA (Geisser–Greenhouse correction) with Holm–Sidak’s multiple comparisons test.DBasal ECAR per µg of protein of B16-F10 cells assayed in glucose (25 mM glucose, 2 mM glutamine) medium using the Seahorse technology.E–GExtracellular lactate (E), extracellular (F) and intracellular (G) glucose levels from B16-F10 cells detected using 1H-NMR (n = 3) analyzed using a RM one-way ANOVA (Geisser–Greenhouse correction) with Holm–Sidak’s multiple comparisons test.H 13C incorporation in intracellular lactate from B16-F10 cells fed with 13C-labeled glucose for 24 h and detected using GC-MS (n = 7). The graph shows the percentage of lactate detected without any carbon isotopically labeled (m0), with one (m0+1), two (m0+2), or three (m0+3) carbons labeled. m0 and m0+3 conditions were analyzed using Friedman’s test with Dunn’s multiple comparisons test.I Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb, and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n = 4), and they were analyzed with a one-sample t-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test. Data information: All quantitative data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 when compared against shCntl. #P < 0.05, ##P < 0.01, when comparing shBNIP3 against shATG5. Unless otherwise specified, each graph represents n = 4 biologically independent experiments and it was analyzed using an RM one-way ANOVA (Geisser–Greenhouse correction) with Holm–Sidak’s multiple comparisons test.