Fig. 3
Mst1/2 sense phagosomal and mitochondrial ROS. a Flow cytometry analysis of cellular ROS levels in WT BMDMs pretreated with or without NAC, infected with E. coli (MOI: 100) and stained with CellRox for 30 min. b SIM of Mst1 staining (red) and DAPI-stained nuclei (blue) in WT BMDMs infected with GFP-E. coli (green) treated with or without NAC as indicated; ×25 magnification of areas outlined in the main images are shown next to the main images. Scale bars, 20 μm. c Immunoblot analysis of phosphorylated (p)-Mob1, Mob1, p-Mst1/2, Mst1, Mst2, and GAPDH in BMDMs pretreated with PBS or NAC (5 μM) and then infected with E. coli (MOI: 100). d Immunoblot analysis of Mst1, Mst2, β-actin and Hsp60 in the cytoplasmic (Cyto) and mitochondrial (Mito) fractions of NAC-treated or non-treated BMDMs infected with E. coli (MOI: 100) for the indicated time. e SIM of Mst1 staining (red), Tomm20 (green) and DAPI-stained nuclei (blue) in WT BMDMs treated with DMSO or antimycin A, with or without NAC pretreatment, as indicated; ×49 magnification of areas outlined in the main images are shown next to the main images. Scale bars, 20 μm. f, g Immunoblot analysis of Mst1, Mst2, β-actin, and Hsp60 in the cytoplasmic (Cyto) and mitochondrial (Mito) fractions of WT BMDMs treated with antimycin A (f) or rotenone (g), with or without NAC pretreatment, for the indicated time. h, i Immunoblot analysis of p-Mob1, Mob1, p-Mst1/2, Mst1, Mst2, and GAPDH in BMDMs treated with antimycin A (h) or rotenone (i) for the indicated time or with antimycin A (h) or rotenone (i) at the indicated dose for 30 min. j, k Immunoblot analysis of p-Mob1, Mob1, p-Mst1/2, Mst1, Mst2, and GAPDH in PBS or NAC pretreated BMDMs followed with 10 μM antimycin A (j) or 3 μM rotenone (k) treatment. l Flow cytometry analysis of mitochondrial ROS levels in THP1 cells treated with 10 μM antimycin A or 3 μM rotenone followed with or without Mst1/2 kinases inhibitor, XMU-MP-1, treatment for indicated times. Data are from one experiment representative of three independent experiments with similar results
