Supplementary Information Notch deficiency decreases hepatic lipid accumulation by induction of fatty acid oxidation No-Joon Song,#, Ui Jeong Yun,#, Sunghee Yang, Chunyan Wu, Cho-Rong Seo, A-Ryeong Gwon,, Sang-Ha Baik, Yuri Choi, Bo Youn Choi, Bahn Gahee, Suji Kim, So-Mi Kwon, Jin Su Park, Seung Hyun Baek, Tae Joo Park, Keejung Yoon 6, Byung-Joon Kim, Mark P. Mattson 7, Sung-Joon Lee, Dong-Gyu Jo,, Kye Won Park, Department of Food Science and Biotechnology, Sungkyunkwan University, Korea. School of Pharmacy, Sungkyunkwan University, Korea. Department of Biotechnology, Graduate School of Life Sciences & Biotechnology, BK-PLUS program, Korea University, 6-7 Seoul Korea. Department of Internal Medicine, Graduate School of Medicine, Gachon University of Medicine and Science, School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, 6 Department of Genetic Engineering, Sungkyunkwan University, Korea. 7 Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, Maryland, USA # These authors contributed equally. Corresponding authors: Dong-Gyu Jo, PhD. School of Pharmacy, Sungkyunkwan University, Suwon -76, Korea. Phone: (+8)--9-778, jodg@skku.edu; Kye Won Park, PhD. Department of Food Science and Biotechnology Sungkyunkwan University, Suwon -76, Korea. Phone: (+8)--9-78, kwpark@skku.edu Supplementary Information: Figure S Figure S Figure S Figure S Figure S Figure S6 Figure S7 Figure S8 Original blots
A C Body weight (g) Insulin (pmol/l). 7 8. B C7 C7 D C7 (g/day) C7 HFD ND 6 7 8 (Weeks) Glucagon (pmol/l)..8. C7 Figure S. Body weight, food intake, insulin, and glucan levels in Notch insufficient mice. (A-C) Body weight (A), food intake (B), insulin (C), levels were not different in high fat diet fed (HFD) C7BL/6 mice (C7) and Notch antisense transgenic () mice. (D) Plasma glucagon level was decreased in mice (n= of each group). Statistically significant differences in the control normal chow fed C7BL/6 (C7-ND) and Notch antisense transgenic () mice or high fat diet fed C7BL/6 mice (C7-HFD) and high fat diet fed Notch antisense transgenic mice (-HFD) were determined using Student s t-test ( P <.).
Free fatty acid(ueq/l) 6 8 6 8 ND HFD ND HFD C7 Figure S. Serum free fatty acid levels from normal diet (ND) or high fat diet fed (HFD) C7BL/6 mice (C7) and Notch antisense transgenic () mice were determined. Statistically significant differences in the control normal chow fed C7BL/6 (C7-ND) and Notch antisense transgenic () mice or high fat diet fed C7BL/6 mice (C7-HFD) and high fat diet fed Notch antisense transgenic mice (-HFD) were determined using Student s t-test ( P <.).
A DAPT - + + - - DBZ - - - + + HES B 8 6 EN GVP reporter DMSO DAPT DBZ Figure S. Notch inhibitors decrease Notch activation. (A) Western blot analysis of HES from DAPT and DBZ treated HepG cells. HepG cells were treated with Notch signaling inhibitors DAPT or DBZ ( µm) and of HES was measured by western blot analysis. (B) HepG cells transfected with the CSL luciferase reporter (notchspecific reporter) and an vector coding for ΔEN (γ-secretase cleavage sites fused to GAL-VP6) were treated with DAPT or DBZ ( µm) and luciferase activity was measured. Data are expressed as the means ± SEM. Statistically significant differences in gene was determined relative to the control by the Student s t-test ( P <.).
Kda C7 Acc Figure S. Western blot analysis of Acc protein in livers from control (C7) and Notch antisense transgenic () mice. Hepatic Acc protein levels were not different in high fat diet fed control and mice.
A Kda C7 Notch Normalized mrna Normalized mrna B... Pparα Acsl C7 mice Cpta Ucp P=.8 Figure S. Notch deficient mice induce of oxidative genes in epididymal white adipose tissues. (A) Reduced Notch in epididymal white adipose tissues from high fat diet fed C7BL/6 mice (C7) and Notch antisense transgenic () mice. Notch protein was measured by immunoblotting. (B) Oxidative genes including Pparα, Acsl, Cpta, and Ucp in epididymal fats of HFD control (C7BL/6) and mice were measured by real time PCR (n= of each group). Data shown represent the mean ± SEM. Statistically significant differences in gene was determined relative to the control (C7BL/6) mice by the Student s t- test ( P <.).
Normalized mrna Acot Nicd Acot Acsl Acsl Ucp Nicd Acox Acox Pparα Ucp Pparα pbp NICD pbp NICD pbp NICD pbp NICD pbp NICD pbp NICD T-L CHT/ T-L CHT/ T-L CHT/ Figure S6. Notch gain of function suppressed the of oxidative genes in preadipocytes. T-L and CHT/ preadipocytes were infected with retrovirus expressing NICD (pbp-nicd ) or virus harboring pbabe-puro (pbp) empty vector and stable cells were selected with puromycin ( µg/ml) for weeks. Gene in stable preadipocytes was measured by real time PCR. Data were expressed as mean ± SEM. Statistically significant differences in gene was determined relative to the control by the Student s t-test (p <.; p <.; p <.).
Normalized mrna C A Lipid accumulation pbp Fabp... pbp pbp-nicd 6 (day) CHT/ pbp- NICD (CHT/) Normalized mrna 8 6 Pparγ Cd6 6 (day) Normalized mrna B D Lipid accumulation pbp Fabp.. T-L pbp pbp-nicd 6 (day) Normalized mrna 6 pbp- NICD (T-L) Pparγ Cd6 6 (day) pbp pbp-nicd pbp pbp-nicd Song et al., Fig S7
Figure S7. Notch gain of function increases lipid accumulation and induces of adipocyte markers during adipocyte differentiation of CHT/ and T-L cells. (A) CHT/ cells were infected pbabe-puro (pbp) or pbabe-nicd harboring retrovirus (pbp-nicd) and stable cells were selected with puromycin ( µg/ml) for week. Stable cells were induced into adipocytes and lipid accumulation on day 6 was assessed by Oil red O staining. (B) T- L cells were infected with pbabe-puro (pbp) or pbabe-nicd harboring retrovirus (pbp-nicd) and stable cells were selected with puromycin ( µg/ml) for week followed by adipocyte differentiation for 6 days. Stable cells were induced into adipocytes and lipid accumulation on day 6 was assessed by Oil red O staining. Gene was measured by real time PCR. (C,D) Stable cells were induced into adipocytes and lipid accumulation on day 6 was assessed by Oil red O staining followed quantification. Lipid accumulation was quantified by measuring the extracted Oil red O dye at nm. Data are expressed as the means ± SEM. Statistically significant differences in gene was determined relative to the control by the Student s t-test ( P <.; P <.; P <.).
Normalized mrna 6 Nicd ctrl NICD 8 Pparα Acsl Acot Acox 6 ctrl NICD ctrl NICD ctrl NICD ctrl NICD Figure S8. Notch gain of function suppressed the of oxidative genes in mature adipocytes. CHT/ preadipocytes were differentiated into mature adipocytes for 8 days and infected with lentivirus expressing NICD (NICD) or control plasmid. Gene in NICD and control cells was measured by real time PCR. Data were expressed as mean ± SEM. Statistically significant differences in gene was determined relative to the control by the Student s t-test (p <.).
Original blots Notch Fig. B p-irs- Fig. F Notch Fig. 6C p-akt Notch Fig A Akt