Quantification and Localization of Sphingolipid Signaling in Aortic Atherosclerosis

Aortae from the origin to the aortoiliac bifurcation were harvested. Spread aortae and cross-sections of aortic sinus and abdominal aorta were stained with ORO. The ORO-positive area over total en face are in each aorta was quantified using ImageJ (NIH) software. Immunofluorescent staining of aortic cryosections and macrophages were performed as described previously^{54 (link)} with the following antibodies: SphK1 (rabbit polyclonal, kindly donated by Dr. Yoshiko Banno)^{49 (link)}, SphK2 (Proteintek #17096-1-AP), p62 (#610832, BD Biosciences), LAMP2 (#550292, BD Biosciences), F4/80 (MCA497RT, Abd Serotec), LC3A/B (#12741, Cell signaling technology (CST)), LC3 mAb (#M186-3, MBL (Nagoya, Japan)), EEA1 (#610456, BD Biosciences), Rab7 (R8779, Sigma Aldrich). Cell nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) (#D1306, Molecular Probes). Stained specimens were observed by confocal fluorescence microscope (LSM 510 Pascal; Carl Zeiss) and quantified by using ImageJ or ZEN 2.1 software (Carl Zeiss).

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Ishimaru K., Yoshioka K., Kano K., Kurano M., Saigusa D., Aoki J., Yatomi Y., Takuwa N., Okamoto Y., Proia R.L, & Takuwa Y. (2019). Sphingosine kinase-2 prevents macrophage cholesterol accumulation and atherosclerosis by stimulating autophagic lipid degradation. Scientific Reports, 9, 18329.

Publication 2019

ImageJ LAMP2 LC3A/B 4′6-diamidino-2-phenylindole (DAPI) LSM 510 Pascal ZEN 2.1

Abdominal aorta Antibodies Aortae Aortic Aortic sinus Cell nuclei Cryosections Face Fluorescence microscope Immunofluorescent Lamp2 Macrophages Molecular probes Rabbit Sphk2

Corresponding Organization : Kanazawa University

Other organizations : Tohoku University, The University of Tokyo, Tohoku Medical Megabank Organization, Ishikawa Prefectural Nursing University, National Institute of Diabetes and Digestive and Kidney Diseases

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Variable analysis

independent variables

Aortae from the origin to the aortoiliac bifurcation were harvested

dependent variables

ORO-positive area over total en face in each aorta was quantified using ImageJ (NIH) software
Immunofluorescent staining of aortic cryosections and macrophages were performed
Stained specimens were observed by confocal fluorescence microscope (LSM 510 Pascal; Carl Zeiss) and quantified by using ImageJ or ZEN 2.1 software (Carl Zeiss)

control variables

Spread aortae and cross-sections of aortic sinus and abdominal aorta were stained with ORO
Antibodies used for immunofluorescent staining: SphK1, SphK2, p62, LAMP2, F4/80, LC3A/B, LC3 mAb, EEA1, Rab7
Cell nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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