Supplementary Figure A 8 SREBPc 6 5 FASN ELOVL6.5.5.5 ACC.5.5 CLOCK.5.5 CRY.5.5 PPARα.5.5 ACSL CPTα.5.5.5.5 MCAD.5.5 PEPCK.5.5 G6Pase 5.5.5.5 BMAL.5.5 Reverbα.5.5 Reverbβ.5.5 PER.5.5 PER B Fasted Refed psrebp nsrebp Supplementary Figure. is increased by feeding (A and B) C57BL/6 mice were fasted for hours and then refed for hours. Both fasted and refed mice were sacrificed at ZT.In the liver, levels of the mrna (A) and protein (B) were determined using qrt-pcr with normalization to TBP mrna levels and western blotting, respectively. Data are represented as mean ±SD, N= for each group. P<.5, P<. (Student s t-test).
Supplementary Figure A ZT ZT Fasted Refed Fasted Refed psrebp nsrebp B 5 SREBPc PEPCK.5.5 G6Pase FOXO Fasted Refed.5.5.5.5.5 CRY.5.5 BMAL.5.5 CLOCK 5 5 Reverbα 8 6 Reverbβ 8 6 PER 6 PER Supplementary Figure. is increased by feeding regardless of time periods (A and B) C57BL/6 mice were fasted for hours and then refed for hours. Both fasted and refed mice were sacrificed at ZT and ZT. In the liver, the levels of protein (A) and mrna (B) were determined using western blotting and qrt-pcr with normalization to TBP mrna levels, respectively. Data are represented as mean ±SD, N= for each group. P<.5, P<., P<. (Student s t-test).
Supplementary Figure A MONKEY COW SHEEP HUMAN RAT MOUSE -75 E-BOX MONKEY COW SHEEP HUMAN RAT MOUSE -5 SRE SRE +5 MONKEY COW SHEEP HUMAN RAT MOUSE SRE +85 B Relative luciferase unit (RLU) 8 6 N.S. MOCK SREBPc Supplementary Figure. promoter analysis usingluciferase assay (A) SRE motifs and E-BOX sequences in the promoter from various species. (B) HEK9T cells were co-transfected with a reporter plasmid containing the WT or E-BOX mutant mouse promoter along with expression plasmids encoding either MOCK or SREBPc. The values represent the mean ± SD (N= for each group). P<.5, P<. (Student s t-test).
Supplementary Figure A 8 6 Ad-MOCK Ad-SREBPc B Ad-MOCK Ad-SREBPc 5 5 5.5..5..5. 5 5 Supplementary Figure. Gene expression profiles (A) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-SREBPc in Figure A and B. The levels of the SREBPc mrna were determined using qrt-pcr with normalization to TBP mrna levels. Data are represented as mean ±SD, N= for each group. P<.5, P<., P<. (Student s t-test). (B) C57BL/6 mice were infected with adenoviruses encoding either MOCK or SREBPc (adenoviral dose of 5 9 viral particles per mouse) through the tail vein in Figure B. Mice were sacrificed 5 days following the injection of adenoviruses and relative mrna levels were determined by qrt-pcr and normalized to the TBP mrna levels. Data are represented as mean ±SD, N=- for each group. P<.5, P<., P<. (Student s t-test).
Supplementary Figure 5 A levels. Relative mrna..8.6... sicon si B 9 6 Ad-MOCK Ad- C...8.6.. G6Pase N.S..5.5.5 8 6 SREBPc Ad-MOCK Ad-SREBPc Supplementary Figure 5. Gene expression profiles (A) HIIE cells were transfected with sicon or si as shown in Figure G. Relative mrna levels were determined by qrt-pcr and normalized to cyclophilin mrna levels. Data are represented as mean ±SD, N= for each group. P<. (Student s t-test). (B) Mouse primary hepatocytes were infected with Ad-MOCK or Ad- in Figure H. Relative mrna levels were determined by qrt-pcr and normalized to TBP mrna levels. Data are represented as mean ±SD, N= for each group. P<. (Student s t-test). (C) Mouse primary hepatocytes isolated from +/+ and -/- mice were infected with Ad-MOCK or Ad-SREBPc in Figure K. Relative mrna levels were determined by qrt-pcr and normalized to TBP mrna levels. Data are represented as mean ±SD, N= for each group. P<. (Student s t-test).
Supplementary Figure 6 A B SREBPc +/+ Ad-MOCK..5..5..5. Ad-Mock Ad-Mock Ad- SREBPc SREBPc SREBPc -/- SREBP +/+ SREBPc -/- -/- Ad-MOCK Ad- SREBPc +/+, Ad-MOCK SREBPc -/-, Ad-MOCK SREBPc -/-, Ad- psrebp FOXO Supplementary Figure 6. overexpression reduces PEPCK mrna and FOXO protein levels in SREBPc -/- mice (A and B) SREBPc +/+ mice injected with Ad-MOCK and in SREBPc -/- mice injected with either Ad- MOCK or Ad- in Figure L and M. In the liver, levels of the mrna (A) and protein (B) were determined using qrt-pcr with normalization to TBP mrna levels and western blotting, respectively. Data are represented as mean ±SD, N=7~ for each group. P<.5, P<. (Student s t-test).
Supplementary Figure 7..5..5..5. SREBPc +/+ SREBPc -/- Supplementary Figure 7. Gluconeogenic gene expression is elevated in SREBPc -/- mice In the liver, the levels of the G6Pase and PEPCK mrna were determined using qrt-pcr with normalization to TBP mrna levels. Data are represented as mean ±SD, N=~ for each group. P<.5 (Student s t-test).
Supplementary Figure 8 DAPI GFP FLAG-MDM Merged GFP- WT GFP- NLS Supplementary Figure 8. MDM subcellular localization in WT and cytosolic overexpressing cells FLAG-MDM was co-transfected with GFP-WT or GFP-cytosolic ( NLS ) in HEK9T cells. Immunocytochemical analysis of FLAG-MDM and GFP was carried out. DAPI,,6-diamidino--phenylindole. Scale bars= µm.
Supplementary Figure 9 A SREBPc G6Pase PEPCK FOXO.5.5.5.5.5.5.5.5.5 B NCD HFD nsrebp FOXO Supplementary Figure 9. Expression profiles of various genes in the liver of NCD and HFD fed mice (A and B) Eight-week-old C57BL/6 mice were fed a NCD or HFD for 8 weeks and sacrificed at ZT. Hepatic gene expression levels (A) were determined by qrt-pcr and normalized by the level of the TBP mrna. N=5 in each group. Hepatic protein levels (B) were analyzed by western blotting.
Supplementary Figure A db/+ db/db ZT: 6 8 6 8 FOXO nsrebp B SREBPc G6Pase PEPCK CRY db/+.5.5 5 8 6 5 5 5 5 5 5 5 5 5 5 Time(ZT) Time(ZT) Time(ZT) Time(ZT) Time(ZT) db/db 8 6 Reverbβ Reverbα.5.5 CLOCK.5.5 5 5 5 5 5 5 5 5 5 5 5 5 Time(ZT) Time(ZT) Time(ZT) Time(ZT) Time(ZT) Time(ZT) BMAL PER 8 6 PER Supplementary Figure. Diurnal gene expression profiles of db/+ and db/db mice liver (A and B) Ten-week-old male db/+ and db/db mice were sacrificed at ZT, 6,, and 8. Protein levels (A) were determined with western blotting. The relative mrna levels of various hepatic genes (B) were determined by qrt-pcr analyses and normalized to the TBP mrna level. Data are represented as mean ±SD, N= for each group.
Supplementary Figure A 5 5 5 G6Pase Control Forskolin Control db-camp PEPCK B G6Pase PEPCK 5 5 5 5 5 5 8 6 C.5.5.5 Control G6Pase Dexamethasone PEPCK Supplementary Figure. Gluconeogenic gene expression is partially attenuated by overexpression upon incubation with forskolin, db-camp, and dexamethasone (A, B and C) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-. The cells were treated with µm forskolin (A), 5 µm db-camp (B), and µm dexamethasone (C) or vehicle (Control) for hours. PEPCK and G6Pase mrna levels were determined using qrt-pcr and normalized to the level of the TBP mrna. The values represent the mean ± SD (N= for each group). P<.5, P<., P<. (Student s t-test).
Supplementary Figure SREBPc +/+ SREBPc -/- SREBPc BMAL CRY.....5.5.5.5.....5.5.5.5.... -6 6 8-6 6 8-6 6 8-6 6 8 Time (CT) Time (CT) Time (CT) Time (CT) Reverbβ RORα CLOCK PER 5 5 5....5.5.5....5.5.5... -6 6 8-6 6 8-6 6 8-6 6 8 Time (CT) Time (CT) Time (CT) Time (CT) SCD FASN G6Pase PEPCK.5..... 8...5 6......5...... -6 6 8-6 6 8-6 6 8-6 6 8 Time (CT) Time (CT) Time (CT) Time (CT) Supplementary Figure. Expression profiles of various genes in the liver of SREBPc +/+ and SREBPc -/- mice Livers were isolated every 6 hours from SREBPc +/+ and SREBPc -/- mice fed with normal chow diet and kept under hours: dark, hours: dark cycle for week. were determined by qrt-pcr and normalized by the level of the TBP mrna. N=- at each time point.
Figure E - - 5- G6Pase - - SREBPc (FLAG) Figure B - - nsrebp - - - FOXO Figure A - FOXO - Figure D - FOXO Figure E - nsrebp psrebp - - FOXO Figure G Figure B - nsrebp - psrebp Supplementary Figure
- pakt - AKT - pfoxo FOXO - Figure 5A - - pakt - AKT FOXO - pfoxo - Figure 5C - - pakt - AKT pfoxo FOXO - Figure 5F
- - - - FOXO (Short exposure) (Long exposure) Figure 5I FOXO - - Figure 6A Figure 6B Figure 6C - (GFP) - - FOXO - - - - 5- (GFP) FOXO IP Input - - Ub- FOXO (HA) - - FOXO
Figure 6D FOXO FOXO - GAPDH - POL II Nucleus Cytosol Figure 6E - UbnFOXO (HA) - nfoxo - FOXO - - - - MDM - Figure 7A - - - - - - IP (GFP) MDM (FLAG) nfoxo - nfoxo - - - MDM (FLAG) (GFP) Input Figure 7B
- - - - - - - - UbnFOXO (HA) nfoxo nfoxo MDM (FLAG) Figure 7C IP Input - - - - - - - IP Input UbnFOXO (HA) nfoxo nfoxo MDM (FLAG) Figure 7C
Figure 8H - - nsrebp FOXO MDM FOXO - Figure 8K nsrebp FOXO - - Figure 8B Figure 8F FOXO - psrebp nsrebp - - Supplementary figure B
Supplementary figure A Supplementary figure 6B - psrebp - psrebp nsrebp - FOXO - - Supplementary figure 9B Supplementary figure 9B nsrebp FOXO FOXO nsrebp - - Supplementary Figure. The original full immunoblot presented in main figures and supplementary figures
Supplementary Table. Sequence of primers for q-rt-pcr Primer Sequences mrsrebpc mr mrpepck mrg6pase hmrfoxo hmfasn mbmal mclock mper hmrcry mscd melovl6 mtbp mper mreverbα mreverbβ Sense 5 -GGAGCCATGGATTGCACATT Antisense 5 -CAGGAAGGCTTCCAGAGAGG Sense 5 -CGTTTGGAAAGGCATTTGG Antisense 5 -CTTCCATTTTGTCAAAGCGTG Sense 5 -AGCCTTTGGTCAACAACTGG Antisense 5 -TGCCTTCGGGGTTAGTTATG Sense 5 -ACACCGACTACTACAGCAACAG Antisense 5 -CCTCGAAAGATAGCAAGAGTAG Sense 5 -CCAAGGCCATCGAGAGC Antisense 5 -GATTGAGCATCCACCAAGAACT Sense 5 -GCTGCGGAAACTTCAGGAAAT Antisense 5 -AGAGACGTGTCACTCCTGGACTT Sense 5 -AACCTTCCCGCAGCTAACAG Antisense 5 -AGTCCTCTTTGGGCCACCTT Sense 5 -TTGCGTCTGTGGGTGTTG Antisense 5 -TGCTTTGTCCTTGTCATCTTCT Sense 5 -TGTGCGATGATGATTCGTGA Antisense 5 -GGTGAAGGTACGTTTGGTTTGC Sense 5 -GGAGCTGCCCAAGAAGC Antisense 5 -AGTAGAAGAGGCGGCAGGA Sense 5 -CCGGAGACCCCTTAGATCGA Antisense 5 -TAGCCTGTAAAGATTTCTGCAAACC Sense 5 -TGCCATGTTCATCACCTTGT Antisense 5 -TACTCAGCCTTCGTGGCTTT Sense 5 -GGGAGAATCATGGACCAGAA Antisense 5 -CCGTAAGGCATCATTGGACT Sense 5 -GGGAGCTCAAACTTCGACTG Antisense 5 -TCGGATGTGATATGCTCCAA Sense 5 -CTGGAGGGCTGCAGTATAGC Antisense 5 -TATTGGAGTCCAGGGTCGTC Sense 5 -TCCTCTAGCTCTGCCTCCAG Antisense 5 -TGGTTTTGCCTGTTTTCACA (m: mouse, h: human, r: rat)
Supplementary Table. sirna Sequences Primer Rat FOXO Mouse Mouse MDM Negative Control Sequences 5 -GAAUGAAGGAACUGGGAAA 5 -CCUCGCAACUGAAGUUGGU 5 -CAGAGAAUGAUGGUAAAGA 5 -CCUACGCCACCAAUUUCGU