Supplemental Data Tamoxifen administration to Vil-Scap- mice.
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- Muriel Potter
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1 Supplemental Data FIGURE S1. Tamoxifen administration to Vil-Scap - mice. In the experiments shown in Fig. 1 to Fig. 5, tamoxifen (2 mg per dose) was dissolved in corn oil and administered by orogastric gavage in staggered time-course fashion (indicated by red arrows) so that tissues were harvested from all mice in a given experiment at the same time. Day 0 mice received corn oil vehicle by gavage in lieu of tamoxifen. Day 1, day 2, day 2.5, day 3, and day 4 mice were exposed to tamoxifen for 1, 2, 2.5, 3, or 4 days, respectively. 1
2 FIGURE S2. Normal small intestine in Vil-Scap - mice at day 2.5 post-tamoxifen. Representative H&E-stained histologic sections from distal small intestine and colon from 13- week-old female Vil-Scap - mice that were administered tamoxifen as described in Fig. S1. Tissues were harvested on day 0 (prior to tamoxifen administration) or on day 2.5 or day 4 after the initial dose of tamoxifen. Small intestine and colon at day 2.5 in Vil-Scap - mice were indistinguishable from day 0 controls. At day 4, severe necrotic injury was seen in small intestine, whereas areas of colon ranged from normal (Day 4, colon, left panel) to mild injury with loss of surface epithelial cells (Day 4, colon, right panel). Magnification 20x. 2
3 FIGURE S3. Crypt-selective disruption of Scap and SREBP proteolysis in Vil-Scap - mice. Scap flox and Vil-Scap - mice (male, 14 weeks of age, 4 mice per group) were administered tamoxifen as described in Fig. S1, and then tissues were collected at day 2.5 after the initial dose. Intestines were scraped with a glass slide to yield upper villi (villus) and then semi-purified crypts were prepared from the scraped mucosa (crypts). Villus and crypt samples were fractionated and aliquots of pooled membrane fraction and nuclear extract (30 μg) were subjected to SDS-PAGE and immunoblot analysis. The precursor and nuclear forms of SREBPs are denoted as P and N, respectively. Asterisks denote nonspecific bands. Scap, SREBP-1, and SREBP-2 are enriched in the crypts relative to villi in control mice and are greatly reduced in the crypts of Vil-Scap - mice. Transgenic, tg. 3
4 FIGURE S4. Inducible whole-body Scap deletion parallels gut-selective deletion. Ligandinduced ubiquitous Scap disruption was accomplished by intercrossing Ubc-CreER T2 transgenic mice with Scap flox mice. Ubc-Scap - (Scap flox/flox ; Ubc-CreER T2 transgenic) developed weight loss, loose stools, and patchy destruction of gastrointestinal mucosa five days after tamoxifen administration. (A) Body weights of and Scap flox and Ubc-Scap - mice (7-8 weeks of age, male, 8 mice per group) that were administered tamoxifen once daily for 4 days as indicated by black arrows. (B) Gross appearance of viscera of a UBC-Scap - mouse on the fifth day after the initial dose of tamoxifen. A yellow arrow denotes a distended, fluid-filled small intestine. (C) Scap and SREBP-2 immunoblots from whole cell extracts of small intestine (30 μg aliquots). The precursor and nuclear forms of SREBPs are denoted as P and N, respectively. (D) H&E-stained histologic sections of proximal small intestine of Scap flox and Ubc-Scap - mice (female, 8-weeks of age) harvested on the fifth day after start of tamoxifen, magnification, 20x. * p <0.05; ** p <0.01 level (Student s t test). 4
5 Supplemental Table S1 Metabolic parameters of Scap flox and Vil-Scap - mice at day 2.5 post-tamoxifen Vil-Scap - Parameter Corn oil Scap flox Vil-Scap - Tamoxifen Tamoxifen Number of mice Body Weight (g) 25.9 ± ± ± 0.8 Intestine weight (g) 1.12 ± ± ± 0.04 Intestine length (cm) 38.2 ± ± ± 0.5 Liver weight (g) 1.26 ± ± 0.04* 1.35 ± 0.05 Total plasma cholesterol (mg/dl) 130 ± ± ± 4 Total plasma triglyceride (mg/dl) 110 ± ± 6 83 ±- 3 Plasma insulin (ng/ml) 1.44 ± ± ± 0.15 Plasma NEFA (mm) 0.28 ± ± ± 0.04 Mice shown here are the same as those used in Figure 4. Each value represents the mean ± SEM of six values. * p <0.05 level (Student s t test) versus Vil-Scap - mice dosed with corn oil. 5
6 Supplemental Table S2 Tissue lipids contents in Scap flox and Vil-Scap - mice at day 2.5 post-tamoxifen Parameter Scap flox Vil-Scap - Number of mice 6 5 Body Weight (g) 21.6 ± ± 0.7 Intestine weight (g) 0.89 ± ± 0.05 IEC total cholesterol (mg/g protein) 25.9 ± ± 1.5 IEC triglyceride (mg/g protein) 87 ± ± 17 Liver weight (g) 1.11 ± ± 0.06 Liver total cholesterol (mg/g tissue) 2.01 ± ± 0.09* Liver triglyceride (mg/g tissue) 10.5 ± ± 1.8 Scap flox and Vil-Scap - mice (female, weeks of age) were administered tamoxifen as described in Fig. S1, and then tissues were collected 2.5 days after the initial dose and isolated small intestinal epithelial cell (IEC) and liver lipid contents were measured. Each value represents the mean ± SEM of five or six values. * p <0.01 level (Student s t test). 6
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Relative Arv1 mrna Adrenal 33.48 +/- 6.2 Skeletal Muscle 22.4 +/- 4.93 Liver 6.41 +/- 1.48 Heart 5.1 +/- 2.3 Brain 4.98 +/- 2.11 Ovary 4.68 +/- 2.21 Kidney 3.98 +/-.39 Lung 2.15 +/-.6 Inguinal Subcutaneous
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