Adiponectin/T-cadherin system enhances exosome biogenesis and decreases cellular
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1 Supplemental Data Adiponectin/herin system enhances exosome biogenesis and decreases cellular ceramides by exosomal release 5 Yoshinari Obata, Shunbun Kita, *,, Yoshihisa Koyama, Shiro Fukuda, Hiroaki Takeda, 4 Masatomo Takahashi, 4 Yuya Fujishima, Hirofumi Nagao, Shigeki Masuda, Yoshimitsu Tanaka, Yuto Nakamura, Hitoshi Nishizawa, Tohru Funahashi,, 5 Barbara Ranscht, 6 Yoshihiro Izumi, 4 Takeshi Bamba, 4 Eiichiro Fukusaki, 7 Rikinari Hanayama, 8 Shoichi Shimada, Norikazu Maeda,, 5 and Iichiro Shimomura
2 Supplemental Figures and Figure legends F FT aorta F FT aorta kda 5kDa 5kDa B C WT serum AKO serum WT serum KDEL KDEL WT serum ATPsF ATPsF WT serum ER ER ER N Go 5 5 Supplemental Figure. Intraluminal accumulation of adiponectin in vesicles of multivesicular bodies (MVBs) in herin-expressing endothelial cells. (A) Comparison of herin protein and accumulated adiponectin in parental endothelial F cells, F cells stably expressing herin (FT cells), and aorta tissue of WT mice. Cells were treated with medium containing 5% WT mouse serum. Equal protein amounts of whole cell lysates were loaded onto SDS-PAGE gels. Ponceau S staining as a loading control is shown in the right panel. n =. Representative results of experiments with similar findings. (B) Confocal immunofluorescence micrographs of FT cells. Cells cultured with WT mouse serum were stained with anti-adiponectin, anti-kdel (endoplasmic reticulum), or anti-atp synthase IF (ATPsF, mitochondria) antibodies. Scale bar, m. Higher magnifications of the indicated areas are shown in the right panels. Scale bar, 5 m. (C) Immunostaining using, -diaminobenzidine tetrahydrochloride (top panels) and immunoelectron micrographs using the pre-embedding immunoperoxidase technique (bottom panels) for adiponectin in FT cells cultured with WT mouse serum or adiponectin knockout (AKO) mouse serum. Bottom right panel: higher magnification of the region outlined in the bottom left panel. Arrowhead, endocytic vesicles; N, nucleus;, mitochondria; ER, endoplasmic reticulum; Go, Golgi body. Scale bar, 4 m (top panels),. m (bottom left panel), and.5 m (bottom right panel).
3 5 Supplemental Figure. Presence of in exosomes from FT cells. Immunoelectron micrograph of exosomes from FT cells, labeled with anti- antibody and visualized by nm-gold conjugated secondary antibody. Scale bar, nm.
4 4 r =.946 p <. Adipoq +/+, +/-, -/- Cdh +/+, +/-, -/- -/- Ad-bGal, Ad Supplemental Figure. Correlation between plasma exosome and. Correlation between plasma exosome and levels in Adipoq littermates (Figure A), Cdh littermates (Figure B), and adiponectin overexpression experiment (Figure C). The mean values of WT controls in each experiment were set to.. Statistical analysis by Pearson correlation test.
5 A serum AKO WT AKO WT B D h h 4h 48h C Tubulin FT cell lysates * *.5.5 6h h 4h 48h ND * * * Supplemental Figure 4. Adiponectin increases exosome production from herin-expressing cells. (A) Western blots of exosome cargos. FT cells were treated with serum from WT or AKO mice. isolated by differential UC were analyzed by western blotting. n =. Representative results of 5 experiments with similar findings. P <. versus AKO serum (unpaired t-test). (B) Non-saturable increase in exosome yields by adiponectin. FT cells were treated with the indicated adiponectin concentrations, and exosomes isolated by differential UC were analyzed by western blotting. The histogram (right) shows relative amounts of and as representatives, respectively. n =. Representative results of experiments with similar findings. (C) Dose-dependent accumulation of adiponectin in FT cells. The histogram (right) shows relative amounts of adiponectin and herin proteins. n =. Representative results of experiments with similar findings. ND, not detected. (D) The time-course analysis of adiponectin effect on exosome production. FT cells were treated with or μg/ml of adiponectin for the
6 65 indicated hours (h), and exosomes isolated by differential UC were analyzed by western blotting. n=. Representative results of experiments with similar findings. *P <.5, P <. versus μg/ml in each incubation time (unpaired t-test). Data are mean ± SEM.
7 A sirna Ctrl ALIX B sirna D Ctrl sirna Ctrl CD8 CD8 sialix ND sisyn ND C sirna Ctrl ALIX ALIX Tubulin DMSO BAPTA Cell lysates DMSO BAPTA-AM.5 ND ND si sicd8 sicd8 E sirna Ctrl Smpd Spr sismpd sispr Relative mrna level sirna Smpd Ctrl Smpd Ctrl Spr Spr 7 Supplemental Figure 5. Association between adiponectin-mediated exosome production
8 75 8 and known machineries for exosome biogenesis. (A-E) FT cells with treated as below were incubated with or μg/ml of adiponectin, and exosome cargo levels were analyzed in exosome pellets by western blotting. n = for each experiment. (A) sirna knockdown of ALIX. (B) sirna knockdown of. (C) treatment with BAPTA-AM (μm). (D) sirna knockdown of, CD8, or CD8. (E) sirna knockdown of neutral sphingomyelinase (Smpd), or sphingosine -phosphate receptor (Spr). Right panel: Relative mrna levels of Smpd and Spr in FT cells transfected with respective sirnas. Data are mean ± SEM.
9 A B C.5 * * * HUVEC CC HepG.5.5 D E Tubulin F G Relative mrna level P-AMPK T-AMPK.5 FT cell lysates sir Adipor sir sir sir+r sir FT sir+r FT cells mock DN P-AMPK/T-AMPK relative ratio mock Relative mrna level sir Adipor sir sir sir sir+r H mock DN.5.5 DN sir+r * mock.5.5 sir ND ND ND * * * sir DN * * 85 9 Supplemental Figure 6. The effect of adiponectin on exosome production is dependent on herin, but not AdipoRs. (A-C) Effect of adiponectin on exosome production in different cells, expressing herin or not. HUVEC (A), CC differentiated myotubes (B), and HepG hepatocytes (C). Each cell type was treated with or μg/ml of adiponectin. n =. Representative results of experiments with similar findings, respectively. *P <.5, *P <. versus μg/ml (unpaired t-test). (D) Relative mrna levels of AdipoR and AdipoR in FT cells transfected with AdipoR sirna (sir) or AdipoR sirna (sir). n=. (E) AdipoRs-knockdown had little effect on adiponectin accumulation
10 95 5 and cellular herin protein. Western blots of FT cell lysates. FT cells transfected with control sirna (), AdipoR sirna, or AdipoR sirna were treated with or μg/ml of adiponectin. The histograms (right) show relative amounts of adiponectin () and herin () proteins. n=. Representative results of experiments with similar findings. ND, not detected. P <. (unpaired t-test). (F) Effect of AdipoRs on exosome stimulation by adiponectin. FT cells transfected with control sirna (), or AdipoR sirna and AdipoR sirna (sir+sir) were treated with or μg/ml of adiponectin, and exosome cargo levels were analyzed in exosome pellets by western blotting. n=. Representative results of experiments with similar findings. P <., *P <. versus μg/ml (unpaired t-test). (G) AMPK phosphorylation. AMPK phosphorylation was significantly suppressed in FT cells stably overexpressing dominant-negative AMPK (DN). Representative results of experiments with similar findings. P <. (unpaired t-test). (H) AMPK activity and exosome stimulation by adiponectin. FT cells stably overexpressing DN were treated with or μg/ml of adiponectin, and exosome cargo levels were analyzed in exosome pellets by western blotting. n=. Representative results of experiments with similar findings. *P <.5; P <.; *P <. versus μg/ml (unpaired t-test). Data are mean ± SEM.
11 A * * mock AC B 6 5 g/ml g/ml Activity (pmol/mg/h) Activity (pmol/mg/h) 4 C 5 ph 4.5 ph 6.5 ph 9. ph Sphingosine (pmol/mg protein) 4 5 Supplemental Figure 7. Ceramidase activities and sphingosine level. (A) Lysates from HEK9 cells transfected with acid ceramidase (AC) expression plasmids showed increased ceramidase activity in acidic ph. Hydrolysis of C-NBD-ceramide was evaluated by reverse-phase HPLC equipped with fluorescence detector. n=. *P <. versus mock (unpaired t-test). (B) Ceramidase activity, determined in lysates from FT cells treated with or μg/ml of adiponectin, under ranges of ph condition. n=. Representative results of experiments with similar findings. There was no statistically significant difference between μg/ml and μg/ml in each ph condition (unpaired t-test). (C) Effect of adiponectin on cellular sphingosine level, determined by liquid chromatography and tandem mass spectrometry, in FT cells. n=. P <. (unpaired t-test). Data are mean ± SEM.
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