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. 2021 Oct 24;9(10):e002875. doi: 10.1136/jitc-2021-002875

Figure 5.

Figure 5

SIRT1 prolongs the survival of Naged by inducing mitophagy. (A) Analysis of the cellular energy phenotype of aged and non-aged neutrophils from the lungs of 2 weeks tumor-bearing mice. (B) The oxygen consumption rate (OCR) of aged and non-aged neutrophils from 2 weeks tumor-bearing mice was measured under basal conditions and in response to treatment with the indicated drugs (1 µM Oligomycin, 1 µM FCCP, and 2.5 µM Rotenone and Antimycin A). (C) The OCR of neutrophils from the BM of naïve mice treated with SRT1720 was measured under basal conditions and in response to treatment with the indicated drugs (1 µM Oligomycin, 1 µM FCCP, and 2.5 µM Rotenone and Antimycin A). (D) Representative TEM images and quantification of the microvesicles (MV) of aged and non-aged neutrophils in PB from patients with breast cancer (left panel) and lung of 2 weeks tumor-bearing mice (right panel). Scale bar, 1 µm (left panel), 0.5 µm (right panel). The white arrow, MV. (E) Levels of the LC3B protein in aged and non-aged neutrophils in the PB from patients with breast cancer and lung of 2 weeks tumor-bearing mice. (F) Levels of the LC3B protein on neutrophils in the PB from patients with breast fibroadenoma and the BM of naïve mice treated with SRT1720 for 4 hours in vitro. (G) Flow cytometry analysis of mitophagy in neutrophils from the PB of patients with breast cancer and lung of 2 weeks tumor-bearing mice, in condition with pepstatin A and leupeptin (inhibitors of autophagy clearance) for 4 hours in vitro. (H) Flow cytometry analysis of mitophagy in neutrophils from the PB of patients with breast fibroadenoma (left panel) and the BM of naïve mice (right panel) treated with SRT1720, in condition with pepstatin A and leupeptin (inhibitors of autophagy clearance) for 4 hours in vitro. (I) Flow cytometry analysis of the survival of aged and non-aged neutrophils from the PB of patients with breast cancer and lung of 2 weeks tumor-bearing mice for 4 hours in vitro. (J) Flow cytometry analysis of the absolute living cells count of aged and non-aged neutrophils from the PB of patients with breast cancer and lung of 2 weeks tumor-bearing mice for 4 hours in vitro. APC labeled CD66b of human samples and Ly-6G of mice samples, neutrophils were presented at the left gate and counting beads at the right). (K) Flow cytometry analysis of the survival of neutrophils from the PB of patients with breast fibroadenoma and the BM of naïve mice treated with SRT1720 in the presence or absence of Mvidi-1 for 4 hours in vitro. Data are presented as the means±SD from one representative experiment. Similar results were obtained from three independent experiments, unless indicated otherwise. Statistical analysis was performed by repeated-measures ANOVA (B, C), two-tailed unpaired Student’s t test (D, G, I), paired Student’s t test (E), one-way ANOVA (F, H, K) and two-way ANOVA (J). *P˂0.05, **p˂0.01, and ***p˂0.001. ANOVA, analysis of variance; APC, allophycocyanin; BM, bone marrow; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; MFI, mean fluorescence intensity; ns, not significant; PB, peripheral blood; TEM, transmission electron microscopy.