2009, 10, 748C754. has been reported to increase resistance against oxidative stress in lens epithelial cells21 and to participate in cancer cell migration PD0166285 and invasion.22 GA also regulates glucose metabolism, inflammation, and cell death in multiple cell lines.23C25 Furthermore, GA can directly bind to and inhibit sumoylation E1 (SAE1/SAE2) enzyme activity.26 In addition, GA has been reported to reduce mitochondrial membrane potential (MMP) and promote mitochondrial damage.27,28 Our previous study showed that genes related to the mitochondria present significant changes in GA-treated mouse bone marrow stromal cells,29 suggesting that GA may play an important role in mitochondria. However, the exact role and mechanism of GA regulation of mitochondrial function are still unclear. Here, we studied the mitochondrial morphology, mitochondrial mass, and mitochondrial function, as well as the underlying mechanisms upon GA (15:1) treatment in HeLa cells. MATERIALS AND METHODS Cell Culture and Treatment HeLa cells were purchased from Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences. ATG7 KO, GFP/Parkin-overexpressing, and control HeLa (gifts from Dr. Quan Chen, Nankai University) cells were maintained in Dulbeccos modified Eagles medium (DMEM) (Gibco, New York, USA, C11995500BT) supplemented with 10% fetal bovine serum (HyClone, Logan, USA, SV30160.03) and 1% penicillin and streptomycin (Gibco, New York, USA, 15140C122). All cells were incubated at 37 C under 5% CO2. In the treatment groups, the cells were treated with 25 or 50 (CST, Boston, USA, 4280; 1:500 dilution), LC3B (Sigma, St. Louis, USA, L7543; PALLD 1:500), and LAMP1 (CST, Boston, USA, 9091; 1:500 dilution) were applied overnight. The cells were then incubated with an anti-rabbit or anti-mouse antibody conjugated with Alexa 488 or Alexa 594 (Jackson ImmunoResearch, Lancaster, USA, AB_2337249 and AB_2307325). After DAPI costaining, the cells were imaged with a Leica TCS SP8 confocal microscope. For colocalization quantification, images were preprocessed by subtraction of a median filter-processed image and background, and then two images were proceeded with ImageJ plugin JACOP (National Institutes of Health).30 Western Blotting Proteins were isolated in ice-cold RIPA buffer (Beyotime, Shanghai, China, P0013B) with proteinase inhibitors, and protein concentrations were determined in bicinchoninic acid assay (BCA). Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto the poly-(vinylidene difluoride) membrane (Millipore, Darmstadt, Germany, IPVH00010), and probed with PD0166285 primary antibodies (Table S1). The protein bands detected by the PD0166285 antibodies were visualized by enhanced chemiluminescence (Beyotime, Shanghai, China, P0018) and evaluated using Quantity One 1D Analysis Software (Bio-Rad, Hercules, CA). mtDNA Measurements Total DNA was isolated by using the Gentra Puregene Cell Kit (Qiagen, New York, USA, 158388) according to the manufacturers instructions. For the measurement of mtDNA copy numbers, the quantitative polymerase chain reaction (qPCR) primers employed for mitochondrial tRNALeu (UUR) and nuclear PD0166285 = 2). GO analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/). Gene expression clustering was analyzed using Cluster 3.0 and visualized using Java TreeView. For gene set enrichment analysis, we applied GSEA v2.2.0 to various functional and/or characteristic gene signatures. Gene sets were obtained from the MsigDB database v3.0. Statistical significance was assessed by comparing the enrichment score to enrichment results generated from 1000 random permutations of the gene set to obtain values (nominal value). MMP Assay The JC-1 probe was used to measure the MMP. Cells cultured in 12-well plates following the indicated treatments were incubated with 500 for 5 min) to remove the cell debris, PD0166285 the pellets were used to determine the protein concentrations via BCA, and the supernatant was added to the substrate solution for the luciferase assay. Luminescence was recorded using an illuminometer with an integration time of 10 s per well. ATP levels were normalized to the protein contents of the samples. Staistical Analysis All experiments were carried out in triplicates. The results are expressed as the mean standard deviation (SD). The level of statistical significance was.