Fig. 4
Dysregulation of Nrf2 in Mst1/2-null macrophages. a RT-qPCR analysis of the mRNA levels of the indicated antioxidant genes in Mst1fl/flMst2fl/fl (WT) and Mst1fl/flMst2fl/fl Lyz-Cre (DKO) BMDMs infected with E. coli (MOI: 100) or treated with LPS, antimycin A or rotenone. b–d Immunoblot analysis of GCLC, GCLM, NQO1, HO-1, p-Mob1, Mob1, Mst1, Mst2, and GAPDH in WT and DKO BMDMs treated with LPS (b), H2O2 (c) or infected with E. coli (MOI: 100) (d). e, f RT-qPCR analysis (e) and immunoblot analysis (f) of the Foxo1, Foxo3, and Nrf2 gene expression in WT B cells, T cells and macrophages. g Confocal microscopy of the distribution of FoxO1, Nrf2, CD3, and F4/80 immunostaining in the indicated colors in spleen sections from WT mice. ×16 magnification of areas outlined in the main images are shown next to the main images. Scale bars, 200 μm. h, i Flow cytometry analysis of ROS levels of FoxO1−, FoxO3− (h) and Nrf2-deficient (i) BMDMs and their WT counterparts. The fluorescence intensity of the ROS dye in the indicated cell samples is shown in the right panel (i). j, k The relative telomere length (T/S ratio) (j) and relative fluorescence intensity of telomere FISH (k) in peritoneal macrophages isolated from 2- or 8-month-old Nrf2+/+ and Nrf2−/− mice. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control (Student’s t test). Data are from one experiment representative of three independent experiments with similar results (mean and s.d. of n = 3 (a, e, experimental replicates); n = 4 (i) or n = 7 (j), biological replicates; mean and s.e.m. of n = 50 (k), experimental replicates)
