Fig. 7

Autophagy restrains minority MOMP in mMKs post IR. a Flow cytometric analysis and quantification of GFP-LC3 expression in BM mMKs of GFP-LC3 mice at indicated hours post IR (hpi) (n = 4). b Flow cytometric analysis and quantification of GFP-mCherry-LC3 expression in Meg-01 cells at indicated hpi (n = 5). c Western blotting analysis and quantification of LC3 lipidation in BM mMKs of mice at 1 dpi (n = 3). d Autophagic clearance of mitochondria in BM mMKs of mice at 1 dpi was assessed by immunofluorescence staining with antibodies specific for LC3 and TOM20. Scale bar = 20 μm. e Flow cytometric analysis and quantification of MMP in BM mMKs of mice with or without HCQ treatment at 1 dpi (n = 5). f Relative mtDNA amounts in cytosols of BM mMKs of mice with or without HCQ treatment at 1 dpi were assessed by qPCR (n = 3). g, h Flow cytometric analysis and quantification of p-TBK1 and p-IRF3 expression in BM mMKs of mice with or without HCQ treatment at 1 dpi (n = 5). i, j Flow cytometric analysis and quantification of IFN-β and p-STAT1 expression in BM mMKs of mice with or without HCQ treatment at 1 dpi (n = 5). k Flow cytometric analysis and quantification of GBP2 expression in BM mMKs of mice with or without HCQ treatment at 3 dpi (n = 5). l MPV in peripheral blood of mice with or without HCQ treatment at indicated dpi (n = 6). m CD62P expression in response to thrombin on washed platelets from mice with or without HCQ treatment at indicated dpi (n = 5). Data represent mean ± standard deviation. ^*P < 0.05, ^**P < 0.01, compared to Ctrl or IR as indicated. Two-tailed unpaired student’s t-test unless stated otherwise. One-way analysis of variance (a, b). ns non-significance, MOMP mitochondrial outer membrane permeabilization, mMK mature megakaryocyte, Ctrl control, IR ionizing radiation, BM bone marrow, hpi hour post IR, dpi day post IR, GFP green fluorescent protein, LC3 microtubule-associated protein 1 light chain 3, LC3-I non-lipidized LC3, LC3-II lipidized LC3, TOM20 translocase of outer mitochondrial membrane 20, MMP mitochondrial membrane potential, HCQ hydroxychloroquine, mtDNA mitochondrial DNA, TBK1 TANK-binding kinase 1, IRF3 interferon regulatory factor 3, IFN-β interferon-β, STAT1 signal transducer and activator of transcription 1, GBP2 guanylate-binding protein 2, MPV mean platelet volume, Cox1 cytochrome c oxidase subunit 1, ND1 NADH dehydrogenase subunit 1, TMRE tetramethylrhodamine ethyl ester, DAPI 4’,6-diamidino-2-phenylindole, GAPDH glyceraldehyde-3-phosphate dehydrogenase