Fig 2: Schematic representation of molecular mechanisms involved in 27HC-induced senescence. 27HC induces a high level of ROS generation, which can downregulate the expression of SIRT1 and subsequently activate the phosphorylation and acetylation of STAT3, which is regulated by SIRT1. Activation of STAT3 triggers aging-related changes in inflammation and the cell cycle leading to neural cell senescence and organism aging.

Fig 3: GluR1/2-β–arrestin1/2–STAT3 complex formation is dependent on GluR1 and GluR2 acetylation.a 293 T cells were transfected with GluR1 or GluR1 plus GluR2. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with GluR2 and Flag antibodies. b 293 T cells were transfected with indicated plasmids followed with or without treatment of glutamate. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with GluR2 and Flag antibodies. c, d 293 T cells were transfected with indicated plasmids. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with Myc and Flag antibodies. e, f Cells were treated with either glutamate or AMPK for different time, cells were subjected to immunoprecipitation with Flag antibody followed by western blot with Myc and Flag antibodies. Data shown are representative of three independent experiments. g, h Mutation assay of acetylation sites that affected interaction between GluR1 and arrestin1/2. 293 T cells were transfected with indicated plasmids and then were treated with or without glutamate. Cells were subjected to immunoprecipitation with Myc antibody followed by western blot with Myc and Flag antibodies. i 293 T cells were transfected with indicated plasmids and then were treated with or without glutamate. Cells were subjected to immunoprecipitation with STAT3 antibody followed by western blot with Myc and Flag antibodies. Data shown are representative of two independent experiments. j 293 T cells were transfected with GluR2-GFP and Stat-RFP. Localization of GluR2 and STAT3 was observed by microscopy. Scale bar: 10 μm. Data shown are representative of three independent experiments.

Fig 4: Glutamate-stimulated translocation of STAT3 into mitochondria.a, b U87 a and HepG2 b cells were treated with glutamate, followed by western blot with indicated antibodies. Data shown are representative of two independent experiments. c 293 T cells were transfected with STAT3 and treated with either calcium or glutamate, or both. Cells were lysed and blotted with indicated antibodies. Data shown are representative of three independent experiments. d C6 cells were treated with or without glutamate and followed by fractionation of cell. Cells were lysed and blotted with indicated antibodies. e C6 cells were treated with glutamate for different time, and then cells were immunostained with anti-STAT3 antibody. Cells were countstained with DAPI. Scale bar: 10 μm. Data shown are representative of two independent experiments. f C6 cells were treated with different combination of glutamate and PhTX-74. Cells were lysed and blotted with indicated antibodies. g C6 cells were treated with different combination of glutamate and PhTX-74. Cells were then lysed and fractionated, and blotted with indicated antibodies.

Fig 5: SH-I-14 represses the proliferation of TNBC cells and reduces acetylation of STAT3A. Structures of carbazole and SH-I-14. B. and C. The anti-proliferative effect of SH-I-14 and CP690550 in TNBC cells. The cells were treated with increasing concentrations of SH-I-14 B. or CP690550 C. for ~72 hr and the viable cells were measured by MTT assay. Data from two independent experiments performed in triplicate are shown as mean ± SEM. D. SH-I-14 induced PARP cleavage in a dose-dependent manner. HS578T cells were treated with increasing concentration of SH-I-14 for 16 hr and the cells (both attached and floating) were harvested for western blot analysis as indicated. β-actin was used as a loading control. E. SH-I-14 increased sub-G1 population of cells. HS578T cells were treated with SH-I-14 for 24 hr and the cells (both attached and floating) were harvested for cell cycle analysis as described in Materials and Methods. F. Effects of SH-I-14 on the level of phospho- and acetyl-STAT3 in HS578T cells. The cells were treated with different concentrations of SH-I-14, AZD1480, or CP690550 for 24 hr and their lysates were subjected to western blot analysis with indicated antibodies. G. SH-I-14 inhibits acetyl-STAT3 in SUM1315MO2 and MDA-MB-231 cells. Cells were treated with increasing amount of SH-I-14 for 24 hr and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control. Abbreviations: Ac, acetyl; and p, phospho; SUM1315, SUM1315MO2.