A-D) Food intake, fecal output, fecal triglycerides and plasma triglycerides were measured in WT and SRC-2 LKO mice (liver specific ablation) fed standard chow ad libitum for 24 hours following an overnight fast (n=5 mice per group).
E-F) Plasma radioactivity and triglyceride levels were measured in chow-fed WT and SRC-2 LKO mice after injection of the lipase inhibitor tyloxapol and gavage with olive oil containing 14C-trioleoylglycerol (n=5 mice per group).
Data are represented as mean + SEM. Two-way ANOVA with Bonferroni posttests to compare replicate means by row was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
G) Intestinal radioactivity levels were measured in WT and SRC-2 LKO mice 2 hours after gavage with olive oil containing 14C-trioleoylglycerol (n=5 mice per group).
Data are represented as mean + SEM. Two-way ANOVA with Bonferroni posttests to compare replicate means by row was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
H-I) Hepatic and biliary BA levels were measured in WT and SRC-2 LKO mice on standard-chow and on 1% cholic-acid diet for two weeks. Bile was obtained from the gall bladder and is representative of intestinal BA levels (n=4-5 mice per group).
J) Fecal triglyceride levels were measured in chow-fed WT and SRC-2 LKO mice upon exposure of mice to a high fat diet (60% calories from fat) and a diet containing high fat plus 1% cholic-acid for 48 hours (n=4-5 mice per group).
Data are represented as mean + SEM. Unless otherwise indicated, unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
See also Fig. S1 and Fig. S2

A) Expression of various BA transporter genes was measured via relative quantitation by QPCR in primary hepatocytes exposed to DMSO or to CDCA from WT and SRC-2 LKO mice. Statistical comparison was performed between WT and SRC-2 KO PHs in each treatment group.
B) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter and the same promoter with a mutated FXRE motif, together with expression plasmids for SRC-2 and FXR and exposed to CDCA. Reporter-gene levels were determined 48 hours after transfection. The empty vector (EV) value was fixed at 1 and the rest of the values are compared relative to that.
C) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter together with expression plasmids for WT SRC-2, SRC-2 mutant with AD1 deletion, SRC-2 mutant with AD2 deletion and SRC-2 mutant with both AD1 and AD2 deletions, and FXR, and exposed to CDCA. Reporter-gene levels were determined 48 hours after transfection. The empty vector (EV) value was fixed at 1 and the rest of the values are compared relative to that.
D) In vivo ChIP assays were performed using liver tissue from WT mice with 150-200 bp amplicons flanking the region containing the FXRE motif of the BSEP promoter and an irrelevant region 3000 bp upstream of the transcription start site. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using two different antibodies that targeted different regions of SRC-2. Control antibody recognized mouse IgG.
E) In vivo ChIP assays were performed using liver tissue from WT and FXR knockout mice with primers flanking the region containing the FXRE motif of the BSEP promoter. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using an SRC-2 specific antibody. Control antibody recognized mouse IgG.
Data are represented as mean + SEM. For all gene expression data unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
See also Fig. S5

A) HepG2 liver hepatoma cells were transfected with pG5Luc (5 Gal4 binding sites driving luciferase expression) together with pBIND SRC-2 (SRC-2:Gal4 DNA binding domain fusion protein) and exposed to 1 mM AICAR. Luciferase levels were determined 48 hours after transfection. The vehicle value was fixed at 1 and the rest of the values are compared relative to that.
B) HepG2 liver hepatoma cells were transfected with pG5Luc together with pBIND SRC-2 and either empty vector (EV) or dominant negative AMPK alpha 2 (DnAMPK) and exposed to 0.3 mM, 0.6 mM and 1 mM AICAR. Luciferase levels were determined 48 hours after transfection. The vehicle value was fixed at 1 and the rest of the values are compared relative to that.
C) HepG2 cell were treated with 1 mM AICAR for 30 min and subjected to immunoprecipitation with an AMPKa2 specific antibody. Immunoprecipated samples were subjected to immunoblotting along with 10% input as indicated.
D) Purified full length SRC-2 protein was subjected to in vitro phosphorylation with or without purified AMPK holoenzyme and AMP as indicated.
E-F) Hepatic BSEP expression and BA content was measured via relative quantitation by QPCR in the liver of WT and AMPK alpha1/alpha2 double knockout (AMPK alpha DKO) mice that were fasted for 24 hours (n = 7-8 mice per group).
G) Hepatic BSEP expression was measured via relative quantitation by QPCR in the liver of mice that were exposed to either PBS or 0.5 mg/g BW AICAR for 12 hours and fed standard chow (n = 6 mice per group).
H) BSEP expression was measured via relative quantitation by QPCR in primary hepatocytes from WT and SRC-2 LKO mice exposed to 1 mM AICAR for 18 hours.
I-J) ChIP assays were performed using HepG2 cells exposed to either vehicle or 1 mM AICAR for 30 min. with primers flanking the region containing the FXRE motif of the BSEP promoter. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 or AMPK alpha 2 occupancy of the BSEP promoter upon chromatin immunoprecipitation, using an SRC-2 or AMPK a2 specific antibody. Control antibody recognized mouse IgG.
All gene expression data are represented as mean + SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
See also Fig. S6

A-D) Food intake, fecal output, fecal triglycerides and plasma triglycerides were measured in WT and SRC-2 −/− mice (whole body ablation) fed standard chow ad libitum for 24 hours following an overnight fast (n=5 mice per group).
Data are represented as mean + SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.

A) Expression of various BA transporter genes was measured via relative quantitation by QPCR in the liver of WT and SRC-2 LKO mice that were exposed to standard-chow or 1% cholic-acid for two weeks (n = 5-7 mice per group).
Data are represented as mean + SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
B) BSEP protein expression was measured via western blot analysis in the liver of WT and SRC-2 LKO mice that were exposed to standard-chow (n=2 mice per group). GAPDH was used as a loading control.
C-F) SRC-2 LKO mice exposed to adenoviral BSEP (Ad. BSEP) were compared with SRC-2 LKO and WT mice exposed to empty adenovirus (Ad. Empty) using tail-vein infusion. 8 days after virus infusion, hepatic BSEP expression, hepatic BA content, fecal triglyceride content and plasma triglyceride content was measured (n=6 mice per group). Statistical comparison was performed between (WT, Ad.Empty) and (SRC-2 LKO, Ad.Empty) and between (SRC-2 LKO, Ad. Empty) and (SRC-2 LKO, Ad.BSEP) groups.
Data are represented as mean + SEM. One-way ANOVA with Tukey's multiple comparison test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.
See also Fig. S3 and Fig. S4

A) BSEP expression was measured via relative quantitation by QPCR in primary hepatocytes exposed to standard doses of the indicated agents for 24 hours.
B) BSEP expression was measured via relative quantitation by QPCR in primary hepatocytes exposed to adenoviruses containing cDNAs representing either constitutively active AMPK alpha or dominant negative AMPK alpha for 24 hours. Statistical comparison was made between the GFP group and either the constitutively active AMPK alpha or dominant negative AMPK alpha groups.
C) BSEP expression was measured via relative quantitation by QPCR in primary hepatocytes exposed to an adenovirus containing cDNA representing dominant negative AMPK alpha and either vehicle or 1mM AICAR for 18 hours.
All gene expression data are represented as mean + SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.

Schematic depicting the cascade that links cellular energy depletion with whole-body energy repletion.