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ATM-induced mitophagy in MCL
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Figure 6. (continued) ATM kinase activity is dispensable in mitophagy in HeLa, mantle cell lymphoma cell lines and primary B-cell lymphomas. (A) Representative flow cytometry (FCS) data showing mitophagy in mantle cell lymphoma (MCL) cell lines (5x106 live cells) treated with FCCP (75 mM for 3 h) or the ATM kinase inhibitor, KU60019 (10 mM for 1 h), either alone or in combination. Following treatments, cells were washed and stained with TMRE (PE) and Mitotracker deep red (APC) and acquired by a FACSCalibur and analyzed with FlowJo software. (B) Relative geometric mean of mitochondrial mass, as in (A), in MCL cell lines treated with FCCP, KU60019 or their combination (mean ± standard error of mean [SEM]; n=7; *P<0.05, **P<0.01: significant difference from respective dimethylsulfoxide [DMSO] or FCCP controls). (C) Representative FCS profile of mitochondrial retention in MCL cell lines treated with FCCP, KU60019 or both in combination, as in (A). (D) FCS analysis showing inhibition of ATM phosphorylation by KU60019 in MCL cell lines, as in (A). Cells treated with neocarzinostatin (NCS) (40 nM for 1 h) served as a positive control. Following treatments, cells were stained with PE-ATMSer1981 and FITC-γH2AXSer139 (as in Figure 2B) and acquired by a FACSCalibur and analyzed with FlowJo software. (E) Immunoblot analysis (30 mg total protein) of MCL cell lines treated with FCCP, KU60019 or their combination, as in (A), and probed with the indi- cated antibodies. NCS treatment served as a positive control for both ATMSer1981 and Kap1Ser824 activation. Total and phosphorylated bands were merged and are shown in color for specificity. Separate blots were cut into pieces and probed with the indicated antibodies. The levels of Parkin, phospho-UBSer65 Parkin and Pink1 protein expression were detected by electrochemoluminescence (ECL). GAPDH was probed as a loading control each time. (F) Densitometry analysis of mitophagy (Tom20 expression) in MCL cell lines treated with the indicated compounds. Assays with FCCP, KU60019 or both in combination (n=7 in Jeko-1 or n=5 in Mino cells) and
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