There have been striking associations of cardiovascular diseases (e. (LOX-1) signaling

There have been striking associations of cardiovascular diseases (e. (LOX-1) signaling to p53 and ensuring cleavage of caspase-3. Additionally, sublethal L5 long-termly inhibited neurite outgrowth in NGF-treated PC12 cells, as evidenced by downregulation of early growth response factor-1 and neurofilament-M. This inhibitory effect was mediated via an interaction between L5 and LOX-1 to suppress NGF-induced activation of PI3k/Akt cascade, but not NGF receptor TrkA and downstream MAPK pathways. Together, our data suggest that L5 creates a neurotoxic stress via LOX-1 in PC12 Ibudilast cells, thereby leading to impairment of viability and NGF-induced differentiation. Atherogenic L5 likely contributes to neurodegenerative disorders. for 10 min and protein concentration in the supernatant was determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CORO1A CA, USA). Cell lysates (40 g/lane) Ibudilast were separated by electrophoresis through 8C12% SDS-polyacrylamide gels and then electroblotted to 0.45 m PVDF membranes (Millipore, Bedford, MA, USA) using a semi-dry transfer apparatus (Hoefer Scientific Instruments, San Francisco, CA, USA). Membranes were blocked in 5% non-fat dry skim milk for 1 h at room temperature, followed by immunoblotting for desired proteins with the assist of specific primary a ntibodies and appropriate HRP-conjugated secondary antibodies. Protein bands developed on the X-ray film were visualized by an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA). Analyses of protein band densities were performed using an Image J software (NIH, Bethesda, MD, USA). 4.9. Statistical Analysis Treatment group means were compared by ANOVA, followed by Dunnetts test or Bonferronis < 0.05). SigmaStat version 4.0 software (Jandel Scientific, San Diego, CA, USA) was used for all statistical analyses. Acknowledgments We are grateful to KMU-LSARC for providing us with LDL particles, including L1, L5, and oxLDL, and excellent technical assistances. This work was supported by grants (MOST 104-2314-B-037-069 owned by Jiz-Yuh Wang and MOST 104-2320-B-570-002 owned by Ching-Tien Lee from the Ministry of Science and Technology, Taipei, Taiwan; and grants (KMU-M106005 owned by Jiz-Yuh Wang and KMU-TP103D02 owned by Chiou-Lian Lai from KMU, Kaohsiung, Taiwan. Abbreviations ADAlzheimers diseaseApoBApolipoprotein BATMAtaxia-telangiectasia mutatedBBBBlood-brain barrierCSFCerebrospinal fluidCVDCardiovascular diseaseDAPI4,6-Diamidino-2-phenylindoleDMEMDulbeccos modified Eagles mediumDSBsDouble strand breaksECEndothelial cellEgr-1Early growth response-1ERKExtracellular signal-regulated kinaseJNKc-JUN NH2-terminal protein kinaseLDLLow-density lipoproteinLDL(?)Electronegative low-density lipoproteinLDL-CLDL-cholesterolLDLRLDL receptorLOX-1Lectin-like oxidized low-density lipoprotein receptor-1MAPKsMitogen-activated protein kinasesMTT3-(4,5-Dimethylthianol-2-yl)-2,5 diphenyltetrazolium bromideNGFNerve growth factorNF-MNeurofilament-mediumOxLDLOxidized LDLPFT-Pifithrin- Author Contributions Jiz-Yuh Wang Ibudilast designed and supervised experiments and wrote the paper. Ching-Tien Lee Ibudilast and Chen-Yen Lin performed experiments and data analysis. Chiou-Lian Lai contributed valuable suggestions and critically revised the manuscript for important intellectual content. Submission of the final manuscript was endorsed by Ibudilast all authors. Conflicts of Interest The authors declare no conflict of interest..