Aims Although oxidized low\density lipoprotein (ox\LDL) in the brain induces neuronal death, the mechanism underlying the damage effects remains largely unknown. cotreatment with rapamycin (an inducer of autophagy) remarkably reversed these effects of ox\LDL. Conclusions Taken together, our results indicated that ox\LDL\induced shift from autophagy to apoptosis contributes to HT\22 cell damage. for 5?minutes. After discarding the supernatants, the collected HT\22 cells were resuspended in 400?L Emixustat binding buffer at a concentration of 10??105 cells/mL. Then, PI and annexin V double\staining apoptosis assay kit was used to quantify apoptotic cells. Apoptosis rate was Keratin 16 antibody assessed by counting the apoptotic cell number per 1??104 cells using flow cytometry (FCM, BD Bioscience). 2.5. Transmission electron microscopy After washing twice with ice\cold PBS, HT\22 cells were fixed with 2.5% glutaraldehyde in 0.15?mM sodium cacodylate at 4C overnight. The cells were postfixed in 2% osmium tetroxide. All samples were dehydrated in ethanol and embedded in epoxy resin. Then, ultrathin sections (70?nm) of adherent cells were performed on an ultramicrotome. The sections were counterstained Emixustat with uranyl acetate and lead citrate and observed using a Jeol JEM SX 100 electron microscope (Jeol, Tokyo, Japan) with images captured. 2.6. Western blot analysis Western blot analyses were performed as described previously.21 Briefly, HT\22 cells were harvested after treatment with indicated agents and washed twice with cold PBS. Total proteins were extracted with a lysis buffer [20?mM Tris\HCL, pH 7.5, 150?mM NaCl, 1% Triton X\100, 1?mM phenylmethylsulphonylfluoride (PMSF), 1?mM Na3VO4, leupeptin, and EDTA] according to the manufacturer’s indications. The samples were centrifuged at 2016 for 10?minutes at 4C, and the supernatant was collected for Western blots. Protein concentration was assessed using a BCA protein assay kit (ComWin Biotech, Beijing, China). Samples were denatured for 5?minutes in boiling buffer. Equal amounts of the boiled proteins (20\30?g per lane) were separated by 10% SDS\polyacrylamide gel electrophoresis (SDS\PAGE). And then, the proteins were transferred to a PVDF membrane and blocked in TBS\T buffer (50?mM Tris\HCl, pH 7.4, 150?mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin (BSA, Sigma) for 2?hours. Subsequently, membranes were incubated overnight at 4C with primary antibodies (anti\LC3\I/II, 1:1000; anti\SQSTML/P62, 1:1000; anti\Bcl\2, 1:1000; anti\Bax, 1:1000; anti\\actin, 1:2000). After washing with TBST for three times, the membranes were incubated with anti\rabbit secondary antibody conjugated to horseradish peroxidase (1:5000) for 2?hours. The bands were visualized using an enhanced chemiluminescence system (ECL, Millipore, Boston, MA, USA). The quantitative analysis of each bolt was carried out by Sigma Scan Pro5 software (San Jose, CA, USA) and normalized to that of \actin. Emixustat 2.7. Statistical analysis Data Emixustat were expressed as mean??SEM, and the significance of intergroup differences was evaluated by one\way analysis of variance (ANOVA: least\significant difference’s test for post hoc comparisons). Differences were considered statistically significant at em P /em .05. 3.?Results 3.1. ox\LDL decreased cell viability of HT\22 cells Considerable researches indicated that ox\LDL can cause neuronal cell death.23, 24, 25 To understand the neurotoxicity of ox\LDL on HT\22 cells, we incubated the HT\22 cells with different concentrations of ox\LDL (0, 12.5, 25, 50, 100?g/mL) or 100?g/mL of nLDL for 24?hours and then detected the cell viability by CCK\8 kit. CCK\8 assay indicated that 100?g/mL of nLDL had no remarkable influence on HT\22 cell viability (Figure?1). However, HT22 cell activity was significantly decreased by ox\LDL in a dose\dependent manner (at the range of 12.5\100?g/mL) with the maximal effect at 100?g/mL (Figure?1). Therefore, 100?g/mL of ox\LDL was used to treat HT\22 cells for 24?hours in subsequent experiments. Taken together, our data suggested that treatment with ox\LDL (100?g/mL) for 24?hours notably lowered the cell viability of HT\22 cells. Open in a separate window Figure 1 Effects of ox\LDL on cell viability in HT\22 cells. HT\22 cells were treated with different concentrations of native LDL (100?g/mL) or ox\LDL (0, 12.5, 25, 50, 100?g/mL) for 24?hours. Cell viability was determined by CCK\8 assay. All the data were shown as mean??SEM of three independent experiments. NS, no significant difference. * em P /em .05, ** em P /em .01 3.2. Autophagy flux was impaired in ox\LDL\induced HT\22 cells Emixustat To investigate the contribution of autophagy to HT\22 cell injury, we first characterized the autophagy changes between control group and ox\LDL\treated group of HT\22 cells by comparing LC3\II and p62 protein levels using Western blot assay. Impaired autophagy can be identified by decreased expression of LC3\II and increased expression of p62 protein. Compared with the control group, LC3\II levels.
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