Figure 4.
ATM inhibitor-induced macropinocytosis leads to increased branched-chain amino acid uptake and affects mTORC1 activity. (A and B) Ovcar8 cells were treated with the ATM inhibitor KU60019 (10 μM) alone or in combination with the indicated metabolites (concentrations in Materials and methods) for 2 h in basal media, and dextran uptake was determined by flow cytometry. n = 3/group, one of at least three experiments is shown. Data represent mean ± SD. Dotted line indicates controls for each metabolite. *P < 0.05 vs. KU60019; one-way ANOVA with Tukey’s multiple comparisons. (C) Ovcar8 cells were treated with the ATM inhibitor KU60019 (10 μM) alone or in combination with the macropinocytosis inhibitor EIPA (25 μM) for 2 h in basal media, and isotopolog enrichment of BCAAs was assessed by mass spectrometry. n = 3/group, one of at least three experiments is shown. Data represent mean ± SD. *P < 0.05 vs. KU60019; one-way ANOVA with Tukey’s multiple comparisons. (D) BCAA metabolite abundance vs. ATM protein expression from DepMap.org. (E) Ovcar8 cells were treated with the ATM inhibitor KU60019 (10 μM) alone or in combination with 200 μM BCAAs for 2 h in basal media, and Western blotting was performed on the indicated proteins. Vinculin was used as a loading control. One of four experiments is shown. (F) Ovcar3 and Ovcar10 cells were treated with the ATM inhibitor KU60019 (10 μM) for 2 h in basal media, and Western blotting was performed on the indicated proteins. Vinculin was used as a loading control. One of three experiments is shown. (G) Fold change (Log2FC) of metabolites in the ascites fluid in the indicated treatment groups.