1. Volume 141, Issue 6, Pages 2166-2175.e7 (December 2011)
Loss of Glucagon-Like Peptide-2–Induced Proliferation Following Intestinal Epithelial Insulin-Like Growth Factor-1–Receptor Deletion 
Katherine J. Rowland, Shivangi Trivedi, Daiyoon Lee, Ken Wan, Rohit N. Kulkarni, Martin Holzenberger, Patricia L. Brubaker 
Gastroenterology 
Volume 141, Issue 6, Pages e7 (December 2011)
DOI: /j.gastro
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2. Figure 1
Inducible tissue-specific deletion of IGF-1R in murine IECs. (A) Two rounds of breeding of villin-Cre-ERT2+/0 and Igf1Rflox/flox mice generated villin-Cre-ERT2+/0; Igf1Rflox/flox (IE-igf1rKO) mice. (B) Recombination was determined by ethidium bromide band intensities obtained by PCR of laser capture microdissection–collected jejunal intestinal epithelial cells from Igf1rflox/flox (control; n = 2) and IE-igf1rKO (n = 2) mice. (C) qRT-PCR of IGF-1R mRNA transcripts (exon boundaries 2–3 and 7–8) from jejunal mucosa of Igf1rflox/flox (n = 7) and IE-igf1rKO (n = 9) mice. *P < .05.
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3. Figure 2
Characterization of IE-igf1rKO mice under basal conditions. Igf1rflox/flox (control) and IE-igf1rKO mice were killed 10 days after tamoxifen induction, followed by examination of (A) SI weight (n = 9 controls; n = 8 KO; representative photomicrographs are shown) and (B) jejunal crypt depths and villus heights (n = 7–9). (C) Positional analysis of Ki67-labeled jejunal crypt IECs from control and IE-igf1rKO mice (n = 8 each). Position 1 designates the cell at the base of the crypt. (D) Quantification of differentiated IEC types in control and IE-igf1rKO jejunum through analysis of synaptophysin-positive (open bars, n = 4), mucin-positive (grey bars, n = 4), and lysozyme-positive (black bars, n = 4) cells. (E) Expression of mRNA transcripts in control (open bars) and IE-igf1rKO (closed bars) mice was determined by qRT-PCR of jejunal mucosa (n = 6–9). Expression was normalized to 18S. (F) Positional analysis of Ki67-labeled jejunal crypt IECs from control (n = 4) and IE-igf1rKO (n = 3) mice after 10-day Long-Arg3–IGF-1 treatment. *P < .05, **P < .01.
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4. Figure 3
β-catenin signaling in response to GLP-2 in IE-igf1rKO mice. (A) Immunofluorescent staining for β-catenin (red), lysozyme (green), and DNA (blue) in jejunal tissue. Quantification of nuclear β-catenin–positivity in non-Paneth crypt cells (indicated by arrows) of control and IE-igf1rKO mice treated with vehicle or GLP-2 (n = 6–8 each). Data are represented as fold of control. Absolute values of nuclear β-catenin–positivity in non-Paneth crypt cells: vehicle control, 1.3; vehicle IE-igf1rKO, 1.3. (B and C) mRNA transcript levels for (B) c-Myc and (C) Sox9, normalized to 18S, in jejunal extracts of control and IE-igf1rKO mice treated with vehicle or GLP-2 (n = 5–10 each). *P < .05.
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5. Figure 4
Weights and morphometric analyses of intestines from fed, fasted, and re-fed IE-igf1rKO mice. Control and IE-igf1rKO mice were fed normally for 24 hours (n = 5–9), fasted for 24 hours (n = 6–8), or fasted for 24 hours and then re-fed for 24 hours (n = 6–11), followed by determination of (A) SI wet weight, (B) jejunal mucosal surface area, and (C) crypt depth and villus height. *P < .05, **P < .01, ***P < .001.
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6. Figure 5
Jejunal crypt–cell proliferation in fed, fasted, and re-fed IE-igf1rKO mice. (A) Positional analysis of Ki67-labeled jejunal crypt IECs from control and IE-igf1rKO mice. Position 1 designates the cell at the base of the crypt. Mice were fed normally (black; n = 7–9), fasted for 24 hours (n = 7–8; blue), or fasted for 24 hours followed by 24 hours of re-feeding (n = 7–11; red). The data shown in panels i and ii are reproduced in panels iii–v to allow direct comparisons between control and IE-igf1rKO mice. For clarity, significance is only reported for fasted vs re-fed groups in panels i and ii. (B) The total incidence of proliferating cells between cell positions 13–20 (area under the curve) for the data shown in panel A. *P < .05, **P < .01, ***P < .001.
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7. Figure 6
Weights and morphometric analyses of intestines from IE-igf1rKO mice after 10-day treatment with GLP-2. Control and IE-igf1rKO mice were treated with vehicle or 0.1 μg/g GLP-2 every 24 hours for 10 days, followed by determination of (A) SI weight (n = 8–10), (B) jejunal mucosal cross-sectional area (n = 7–9), and (C) jejunal crypt depth and villus height (n = 7–10). *P < .05, **P < .01.
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8. Figure 7
Jejunal crypt–cell proliferation in control and IE-igf1rKO mice after 10-day treatment with GLP-2. (A) Positional analysis of Ki67-labeled jejunal crypt IECs from control (solid lines) and IE-igf1rKO (dashed lines) mice. Position 1 designates the cell at the base of the crypt. Mice were administered vehicle (black line; n = 8–9) or GLP-2 (grey line; 0.1 μg/g; n = 8–10) for 10 days after tamoxifen induction. (B) The total incidence of proliferating cells between cell positions 15–20 (area under the curve) for the data shown in panel A. *P < .05, **P < .01, ***P < .001.
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9. Supplementary Figure 1
Characterization of Igf1rflox/flox (control) and IE-igf1rKO mice under basal conditions immediately after treatment with tamoxifen. (A–D) Intestine, (E) jejunal mucosa, and (F) liver. (B, E, F) White bars, control; closed bars, KO (n = 4–15). *P < .05.
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10. Supplementary Figure 2
Representative sections of jejunum from tamoxifen-treated (A) beta-galactosidase/enhanced green fluorescent protein, and (B) villin-Cre-ERT2+/0; beta-galactosidase/enhanced green fluorescent protein mice.
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11. Supplementary Figure 3
mRNA expression in the ileal mucosa of Igf1rflox/flox (control) and IE-igfrKO mice 10 days after tamoxifen induction (n = 5–6). *P < .05.
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12. Supplementary Figure 4
mRNA expression in the ileal mucosa of Igf1rflox/flox (control) and IE-igfrKO mice 1 day after tamoxifen induction (n = 5–7). *P < .05.
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13. Supplementary Figure 5
mRNA expression in the colonic mucosa of Igf1rflox/flox and IE-igfrKO mice 1 day after tamoxifen induction (n = 5). *P < .05.
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14. Supplementary Figure 6
IRS1 mRNA expression in jejunal mucosa of Igf1rflox/flox and IE-igfrKO mice after tamoxifen induction followed by overnight fasting and treatment with vehicle or GLP-2 for 90 minutes (n = 6–8).
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15. Supplementary Figure 7
Body weight (A; n = 6–11) and percentage dry weight (n = 6) of (B) jejunum and (C) ileum from Igf1rflox/flox and IE-igf1rKO mice that were fed normally for 24 hours (closed bars), fasted for 24 hours (open bars), or fasted for 24 hours and then re-fed for 24 hours (grey bars).
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16. Supplementary Figure 8
mRNA transcript levels of (A) IGF-1 and (B) IGF-2 in jejunal mucosa extracts of Igf1rflox/flox and IE-igf1rKO mice treated for 10 days with vehicle (open bars) or GLP-2 (closed bars; n = 7–8). *P < .05.
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17. Supplementary Figure 9
Body weight of Igf1rflox/flox and IE-igfrKO mice treated with vehicle or GLP-2 for 10 days after tamoxifen induction (n = 8–10).
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18. Supplementary Figure 10
Villin-Cre-ERT2+/0 mice were pretreated with tamoxifen or vehicle followed by treatment with GLP-2 or phosphate-buffered saline (PBS) for 10 days. GLP-2 (closed bars or grey lines) or PBS (empty bars or black lines) was administered to vehicle-pretreated (as indicated or dashed lines) or tamoxifen-pretreated (as indicated or solid lines) villin-Cre-ERT2+/0 mice, followed by analysis of (A) SI weight, (B) jejunal growth and (C) jejunal proliferation (n = 4). *P < .05, **P < .01.
Gastroenterology  , e7DOI: ( /j.gastro )
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