Fig. 1

Increased ROS levels and apoptotic events in Mst1/2-null macrophages. a Flow cytometry of Mst1^fl/flMst2^fl/fl (WT) and Mst1^fl/flMst2^fl/fl Lyz-Cre (DKO) BMDMs stained for 30 min with the ROS dye CM-H2DCFDA. Quantification of the relative fluorescence intensity in the indicated cell samples is shown in the right panel. b, c Quantification of protein carbonylation levels (b) and immunoblot analysis of phosphorylated (p)-H2A.X (Ser139), H2A.X, pro- and cleaved-caspase 3, PARP, Mst1, Mst2, and GAPDH (c) in WT and DKO BMDMs pretreated with or without NAC. d Fluorescence microscopy of p-H2A.X staining (red) and DAPI⁻ stained nuclei (blue) in WT BMDMs (left) or DKO DMDMs (right) labeled with CFSE, mixed with unlabeled DKO BMDMs (arrows) or WT BMDMs (stars), and treated with H₂O₂ and NAC as indicated. Scale bars, 20 μm. e, f Annexin V/DAPI staining (e) and quantification of Annexin V⁺DAPI⁻ cells (f) among WT and DKO BMDMs pretreated with or without NAC. g, h Flow cytometry (g) and quantification (h) of the percentage of Annexin V⁺DAPI⁻ cells among WT and DKO CD11b⁺F4/80⁺ cells pretreated with or without NAC. i Fluorescence microscopy of p-H2A.X staining (red) and DAPI-stained nuclei (blue) in WT BMDMs (left) or DKO DMDMs (right) labeled with CFSE, mixed with unlabeled DKO BMDMs (arrows) or WT BMDMs (stars), and treated with DMSO, antimycin A or rotenone. Scale bars, 20 μm. j, k Annexin V/DAPI staining (j) and quantification of Annexin V⁺DAPI⁻ cells (k) among WT and DKO BMDMs treated with DMSO or antimycin A. *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 (f), n = 4 (a, b), n = 5 (k) or n = 8 (h) biological replicates)