Hepatic Gene Expression of Bile Acid Synthesis Genes from Wild-Type and Fxr−/− Mice

A 2.0 Acox2 B 2.0 Akr1c14 C 2.0 Akr1d1 D 2.0 Amacr ** 1.5 1.5 1.5 1.5

1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA

0.0 0.0 0.0 0.0 GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− E F 2.0 Cyp7b1 2.0 Cyp27a1 G 2.0 Cyp39a1 H 2.0 Hsd3b7

1.5 1.5 1.5 1.5 *

1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA

0.0 0.0 0.0 0.0 GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− I J K 2.0 Hsd17b4 2.0 Scp2 2.0 Slc27a5 L Fxr Cyp7a1 Cyp8b1 1.0 1.5 1.5 1.5 *

1.0 1.0 1.0 0.5 *** *** 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA *** *** *** 0.0 0.0 0.0 0.0 *** GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h Time (h) 0 4 16 0 4 16 0 4 16 FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− post plating M N CYP7A1 CYP8B1 Supplementary Figure 1 – FXR activation leads to rapid changes in gene expression 1.0 1.0 (A-K) Hepatic gene expression of bile acid synthesis genes from wild-type and Fxr−/− mice. Mice were treated with either vehicle (WT n=16; Fxr−/− n=7) or GSK2324 and RNA isolated after 30 minutes (WT n=12; Fxr−/− n=6), 1h (WT n=10; Fxr−/− n=6) or 2h (WT n=8; Fxr−/− 0.5 0.5 mRNA half-life n=7). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. (L)

mRNA (Fold Change) mRNA mRNA half-life mRNA (Fold Change) mRNA Gene expression of Cyp7a1, Cyp8b1, and Fxr from primary hepatocytes that were freshly

0.0 0.0 isolated (t=0) or the same hepatocytes plated for 4 or 16 hours on collagen coated plates 012345678 012345678 (n=6 wells/condition). (M,N) CYP7A1 and CYP8B1 mRNA levels in IHH cells treated with Time (h) Time (h) actinomycin D for various time points. Gene expression was determined by RT-qPCR and data are normalized to 36B4. Data are normalized to 36B4. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by one way ANOVA. A 2.0 Cyp8b1 (Liver) D 1.5 Cyp8b1 (Liver)

1.5

1.0 * ** 1.0

* 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA **

0.0 0.0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h GSK − 30’ 1h 2h FXR Fxrflox-flox FxrL-KO Shp−/− 4 B 20 Shp (Liver) E Insig2a (Liver) *** *** ** 3 *** 15 *** *** 10 2

5 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA

0 0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h GSK − 30’ 1h 2h FXR Fxrflox-flox FxrL-KO Shp−/−

C 4 Fabp6/iBabp (Ileum)

3 *** *** ***

2 ** * *

1 mRNA (Fold Change) mRNA

0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h FXR Fxrflox-flox FxrL-KO

Supplementary Figure 2 – Decrease in Cyp8b1 following FXR activation required hepatic FXR but is independent of SHP (A-C) Gene expression of hepatic Cyp8b1 and Shp and ileal Fabp6 in littermate Fxrflox-flox and FxrL-KO mice were treated with either vehicle (n=13 Fxrflox-flox; n=11 FxrL-KO) or GSK2324 for 30 minutes (n=9 Fxrflox-flox; n=8 FxrL-KO), 1h (n=7 Fxrflox-flox; n=9 FxrL-KO), 2h (n=8 Fxrflox-flox; n=7 FxrL-KO) or 4h (n=5 Fxrflox-flox; n=10 FxrL-KO). (D- E) Gene expression of hepatic Cyp8b1 and Insig2a in Shp−/− mice treated with either vehicle (n=8) or GSK2324 for 30 minutes (n=8), 1h (n=7) or 2h (n=8). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by one way ANOVA. A B C CDCA Veh GW D Cyp7a1 Cyp8b1 Shp I.B. 1.5 4 ZFP36L1 1.5 CYP7A1

*** I.B. PDI 3 1.0 2.0 ZFP36L1 1.0Legend

* Legend ** fold fold change) fold fold change) 2 1.5 ( ( Legend 0.5 1.0

1 fold change) 0.5 ( mRNA mRNA mRNA mRNA *** *** *** 0.5 *** 0.0 0 (Fold Change) mRNA mRNA mRNA Diet Ctr CA Ctr CA Diet Ctr CA 0.0 0.0 Diet Diet Diet Treatment CDCA Veh GW4064 Treat. CDCA Veh GW4064 E F G 4 4 ZFP36L1 ZFP36L1 ZFP36L1 Vehicle 1.00 30' 3 3

** 1h 0.75 *** ** 2 2h * 2 * 0.50 ** 4h 1 0.25 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0.00 0 02468 0 Time − 30’ 1h 2h Time − 30’ 1h 2h Time (h) Treat. GW4064 (1µM) Treat. CDCA (100µM)

Supplementary Figure 3 – Zfp36l1 is a direct FXR target gene (A,B) Hepatic Cyp7a1,Cyp8b1 and Shp mRNA expression in C57BL/6 wild-type fed a control diet (Ctr; n=8) or a diet supplemented with 0.5% cholic acid (CA Diet; n=8) for 7 days. Gene expression analysis was determined by RT-qPCR and normalized to Tbp. (C) ZFP36L1 mRNA and protein (D) CYP7A1 mRNA in immortalized human hepatocytes (IHH) treated with either CDCA (100µM) or GW4064 (1µM) for 24h (n=3 wells/condition). (E) ZFP36L1 mRNA levels were measured in IHH cells treated with actinomycin D for the indicated times (n=6 wells condition). (F and G) ZFP36L1 mRNA levels in IHH cells treated with either CDCA (100µM) or GW4064 (1µM) for 30’, 1h and 2h (n=4 wells/condition). Gene expression data was normalized to 36B4 or TBP. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by Student’s t-test for A-D and one-way ANOVA for F-G. A I.B. ZFP36L1

I.B. PDI B C 4 1.5 1.5 Zfp36l1 Cyp7a1 Ad-Ctr Ad-Zfp36l1

*** 1.0 1.0

2 ** ** ** 0.5 0.5 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0 0.0 0.0

Scp2 Acox2 Akr1d1 Amacr Cyp8b1 Cyp7b1 Hsd3b7 Akr1c14 Cyp27a1Cyp39a1 Hsd17b4 Slc27a1 D E F G H I

) 4000 15 1.5 250 25 40 l) M Insulin Glucose ALT AST µ µ

) 200 20 3000 dL 30

* 10 1.0 150 15 2000 20 100 10 ALT (U/L) ALT 5 0.5 (U/L) AST

1000 Insulin (ng/ml) 10 Glucose (mg/ 50 5 Gall Bladder Volume ( Gall Bladder Volume 0 0 0.0 0 0 0 Biliary BAs/gall bladder ( bladder BAs/gall Biliary

Supplementary Figure 4 – ZFP36L1 gain-of-function decreases CYP7A1 and alters bile acids in vivo (A) Zfp36l1 mRNA and protein and (B) Cyp7a1 and (C) other bile acid synthesis genes in female C57BL/6 wild-type mice treated with either Ad-control (Ad-Ctr) or Ad-Zfp36l1 for 5 days (n=6-7 mice/group). (D) Bile acid amount and (E) gall bladder volume (F) plasma insulin (G) plasma glucose (H) plasma alanine aminotransferase (ALT) and (I) plasma aspartate aminotransferase (AST) in male Ad-control (Ad-Ctr) or Ad-Zfp36l1 treated mice (n=10 mice/group). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001) by Student’s t-test. A B C D E F G H 40 15000 60 TAG 50 FFA 20 ALT 50 AST 1.5 Insulin 300 Glucose * ) ** 30 40 40

15 dL 10000 40 1.0 200

) 30 30 l) dL dL M µ ( µ 20 g/

g/ 10 ( m m 20 20 ALT (U/L) ALT 5000 20 (U/L) AST 0.5 100

10 5 Insulin (mg/ml) 10 10 Glucose (mg/ Gall Bladder Volume Biliary BAs/gall bladder BAs/gall Biliary 0 0 0 0 0 0 0.0 0

I J K fl-fl 8000 Zfp36l1 8000 Zfp36l1fl-fl 8000 ) Zfp36l1fl-fl ) L-KO Zfp36l1 L-KO hr Zfp36l1 L-KO hr 6000 6000 6000 Zfp36l1

4000 4000 4000 (ml/kg/ (ml/kg/ 2 2 p=NS 2000 2000 2000

VO p=NS p=NS VCO

0 0 0 Average TotalActivity 7am 8am 9am 1am 2am 3am 4am 5am 6am 7am 8am 9am 1am 2am 3am 4am 5am 6am 7am 8am 9am 1am 2am 3am 4am 5am 6am 7am 8am 9am 1am 2am 3am 4am 5am 6am 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 7am 8am 9am 1am 2am 3am 4am 5am 6am 7am 8am 9am 1am 2am 3am 4am 5am 6am 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 1pm 2pm 3pm 4pm 5pm 6pm 7pm 8pm 9pm 11am 11am 11am 11am 11pm 11pm 10am 12am 10am 12am 11pm 11pm 10am 12am 10am 12am 12pm 10pm 12pm 10pm 12pm 10pm 12pm 10pm 11am 11am 11pm 11pm 10am 12am 10am 12am 12pm 10pm 12pm 10pm

Supplementary Figure 5 – ZFP36L1 loss-of-function alters bile acids in vivo (A) Biliary bile acid amount (B) gall bladder volume (C) plasma triglyceride (TAG) (D) plasma free fatty acids (FFA) (E) plasma alanine aminotransferase (ALT) (F) plasma aspartate aminotransferase (AST) (G) plasma insulin and (H) plasma glucose in littermate male Zfp36l1flox-flox (n=10) and Zfp36l1L-KO mice (n=12). (I) flox-flox L-KO VO2, (J) VCO2 (K) Activity of littermate male Zfp36l1 (n=5) and Zfp36l1 mice (n=6) placed in metabolic cages at 60 days of age fed a standard rodent diet. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01) by Student’s t-test for A-H and two way ANCOVA with repeated measures for I-K. A 30 B C 20 D 30 Zfp36l1fl-fl Zfp36l1fl-fl Zfp36l1fl-fl Zfp36l1fl-fl

L-KO 40 L-KO L-KO L-KO Zfp36l1 Zfp36l1 15 Zfp36l1 Zfp36l1 25 25 * 30 * 10 ** *** *** ** *** * 20 ** *** 20 ** *** * Fat Mass (g) ** Lean Mass (g)

Lean Mass (g) 5 Body Weight (g) Body Weight 20

15 0 15 0 20 40 60 80 0 20 40 60 0 20 40 60 80 0 20 40 60 80 Days on HF Diet Days on West. Diet DaysDays on on HF Diet Diet DaysDays on on HF Diet Diet Days on Diet E F G 4000 p=NS Zfp36l1fl-fl 10000 8000 Zfp36l1fl-fl Zfp36l1fl-fl Zfp36l1L-KO L-KO L-KO ) Zfp36l1 3000 ) 8000 Zfp36l1 hr

hr 6000

6000 2000 4000 (ml/kg/ (ml/kg/ 2

2 4000 1000 VO

VCO 2000 2000 p=NS p=NS

Average TotalActivity 0 9am 1am 5am 9am 1am 5am 9am 1am 5am 0 0 1pm 5pm 9pm 1pm 5pm 9pm 1pm 5pm 9pm 9am 1am 5am 9am 1am 5am 9am 1am 5am 9am 1am 5am 9am 1am 5am 9am 1am 5am 1pm 5pm 9pm 1pm 5pm 9pm 1pm 5pm 9pm 1pm 5pm 9pm 1pm 5pm 9pm 1pm 5pm 9pm H I J K L ) 400 Glucose 80 AST 1.5 5000 0.8 Il1b Tnfa ** dL ** 4000 300 60 0.6 p=0.09 1.0 3000 Combustion 200 40 * 0.4 of /g feces)

** 2000 0.5 cal 100 20 ( 0.2 Plasma AST (U/L) Plasma 1000 Fecal output (g/24h) Fecal mRNA (fold change) mRNA Plasma Glucose (mg/

0 0 0.0 Gross Heat 0 0.0

Supplementary Figure 6 – Loss of hepatic Zfp36l1 results in resistance to diet-induced obesity, protection from steatosis and reduced lipid absorption. (A) Lean mass in grams (g) of male littermate Zfp36l1flox-flox (n=31) and Zfp36l1L-KO (n=19) mice fed a Western diet for 64 days. (B) Body weight in grams (g) (C) fat mass in (g) and (D) lean mass in (g) of male littermate Zfp36l1flox-flox (n=6) and Zfp36l1L-KO (n=8) mice fed a high fat (HF; 45kCal) diet for 77 flox-flox days (n=6-8 mice/genotype). (E) VO2, (F) VCO2 and (G) activity of male littermate Zfp36l1 (n=5) and Zfp36l1L-KO (n=6) mice fed a Western diet for 64 days. (H) Plasma glucose, (I) Plasma aspartate aminotransferase (AST) and (J) hepatic gene expression of Il1b and Tnfa in male littermate Zfp36l1flox- flox (n=12) and Zfp36l1L-KO (n=9) mice fed a Western diet for 64 days. (K) Fecal calorimetry analysis and (L) Fecal output of Zfp36l1flox-flox (n=12) and Zfp36l1L-KO (n=9) mice fed a Western diet for 64 days. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01) by two-way ANOVA with repeated measures for A-D, two-way ANCOVA with repeated measures for E-G and Student’s t-test in H-L. Fig. 3D Fig. 3E

ZFP36L1

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ZFP36L1

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ZFP36L1

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ZFP36L1

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ZFP36L1

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ZFP36L1

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CYP7A1

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