1. Volume 145, Issue 4, Pages 820-830.e10 (October 2013)
Progastrin Stimulates Colonic Cell Proliferation via CCK2R- and β-Arrestin–Dependent Suppression of BMP2 
Guangchun Jin, C. Benedikt Westphalen, Yoku Hayakawa, Daniel L. Worthley, Samuel Asfaha, Xiangdong Yang, Xiaowei Chen, Yiling Si, Hongshan Wang, Yagnesh Tailor, Richard A. Friedman, Timothy C. Wang 
Gastroenterology 
Volume 145, Issue 4, Pages e10 (October 2013)
DOI: /j.gastro
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2. Figure 1
Progastrin down-regulates BMP pathway and Id4 genes in the colonic mucosa of hGAS mice. (A) Immunohistochemistry for Bmp2, pSmad1/5/8, and Id4 protein expression in the colonic mucosa of hGAS and WT mice (original magnification, ×800). Quantitative reverse transcription PCR analysis of (B) Bmp2, (C) Bmp7, and (D) Id4 mRNA levels in the colonic mucosa of hGAS, WT, and GAS−/− mice (n = 6 mice/group). Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. (E) Enzyme-linked immunosorbent assay for Bmp2 protein in the colonic mucosa of hGAS and WT mice (n = 4 mice/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01. (F) Western blot analysis of Bmp2, pSmad1/5/8, Id4 protein levels from murine colonic mucosa (n = 3 mice/group).
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3. Figure 2
Progastrin increases mucosal proliferation through CCK2R-dependent suppression of Bmp2 pathway. (A) Representative pictures (original magnification, ×40 and ×200) and (B) the numbers of colonic crypt organoids from WT and CCK2R−/− mice (n = 3 plates/group) after 3 days in culture in Wnt3A, epidermal growth factor, and R-spondin1 containing media with or without progastrin (1ug/mL). (C) Quantitative reverse transcription PCR analysis of Bmp2 mRNA expression in colonic organoids (n = 3 plates/group). Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. (D) Immunohistochemistry for BrdU in the colonic mucosa after Bmp2 protein injection (original magnification, ×300). (E) The percentage of BrdU-positive cells in colonic crypts (n = 23 crypts/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
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4. Figure 3
Progastrin binds to CCK2R to increase cell proliferation. (A) Immunofluorescence staining of progastrin binding on CCK2R-expressing cells after 30 minutes of progastrin treatment (original magnification, ×800). MTT assay after progastrin treatment on Colo320(+) and Colo320(−) cells (B) or pretreatment with CCK2R antagonist YF476 on Colo320(+) (C) and Colo320(−) cells (D) (n = 6 plates/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01.
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5. Figure 4
Progastrin stimulates proliferation through CCK2R-dependent mechanism. (A) Fluorescence-activated cell sorting analysis of BrdU incorporation after progastrin treatment to the Colo320(+) and Colo320(−) cells. Horizontal axis: BrdU−fluorescein isothiocyanate. (B) Comparison of BrdU-positive cell populations after progastrin treatment of Colo320(+) and Colo320(−) cells (n = 3 plates/group). (C) AGSE cell morphology in serum-free medium with or without YF476 treatment (white arrow: mitotic cells) (original magnification, ×600). (D) Percentage of mitotic events in AGSE cells with or without progastrin treatment (n = 8 yields/group). All values represent the mean ± SD. ∗P < .05.
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6. Figure 5
Progastrin increases cell proliferation through CCK2R-dependent, β-arrestin pathway. (A) Calcium influx in AGSE cells after progastrin and G-17 treatment. MTT assay of Colo320(+) (B) and Colo320(−) (C) cells (n = 6 plates/group), and quantitative reverse transcription PCR analysis of Bmp2 mRNA levels in Colo320(+) (D) and Colo320(−) (E) cells (n = 3 plates/group) after transfection with 200 ng/140 uL β-arrestin 1 and 2 small interfering RNA (siRNA). (F) Percentage of symmetric divisions of BrdU-positive cells after transfection with β-arrestin 1 and 2 siRNA in Colo320(+) and Colo320(−) cells (n = 6 plates/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01.
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7. Figure 6
Progastrin increases cell proliferation through Bmp2 suppression. Quantitative reverse transcription PCR analysis of Bmp2 mRNA expression (n = 3 plates/group), the expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (A), protein levels with enzyme-linked immunosorbent assay (n = 3 plates/group) (B), and Western blot (n = 2 plates/group) (C) in Colo320(+) and Colo320(−) cells after progastrin treatment. MTT assay of Colo320(+) and Colo320(−) cells after Bmp2 protein (n = 6 plates/group) (D) and Noggin treatment (n = 6 plates/group) (E). (F) Quantitative reverse transcription PCR analysis of Id4 mRNA expression levels after progastrin treatment of Colo320(+) and Colo320(−) cells (n = 6 plates/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
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8. Figure 7
Progastrin increases stem or progenitor cell proliferation in AGSE cells. (A) Fluorescence-activated cell sorting analysis of CD44-positive cells in AGSE and AGS cells after progastrin treatment. Horizontal axis: CD44-phycoerythrin (PE). (B) CD44-positive cell populations in AGSE and AGS cells after progastrin treatment (n = 3 plates/group). (C) Immunofluorescence analysis of BrdU-positive cells. Representative examples for symmetric or asymmetric cell divisions are shown (original magnification, ×600). (D) Symmetric and asymmetric cell populations after progastrin treatment to the AGSE cells (n = 6 plates/group). (E) Symmetric and asymmetric cell populations after Bmp2 protein treatment to the AGSE cells (n = 6 plates/group). All values represent the mean ± SD. ∗P < .05.
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9. Supplementary Figure 1
Progastrin decreases Bmp2 protein levels both in vivo and in vitro. Western blot analysis of Bmp2 protein levels in colonic extracts from hGAS and WT mice (n = 3 mice/group) (A), and in Colo320(+) (B) and Colo320(−) (C) cells after progastrin treatment (n = 2 plates/group).
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10. Supplementary Figure 2
Microarray analysis of BMP pathway genes in the colonic mucosa of hGAS, GAS−/−, WT mice. (A) Heat map and hierarchical clustering of all Bmp pathway genes in 3 comparisons for which praw ≤ .05 in hGAS, GAS−/−, WT mice. The colors represent log base 2 fold changes for each comparison: red, up; green, down. (B) Proposed mechanism by which a decrease in Bmp2 leads to an increase in colonic tumorigenesis.
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11. Supplementary Figure 3
Progastrin promotes budding events in colonic organoids. (A) Number of colonic crypts after 7 days in culture (n = 4 plates/group) with or without noggin or progastrin. (B) The number of colonic crypts from ubiquitin C-GFP mice in culture (day 7) after progastrin treatment. Data are shown as mean ± SD. ∗∗P<.01; ∗∗∗P < (C) Immunofluorescence pictures of cultured colonic organoids (day 7) from ubiquitin C−GFP mice with or without 1 ug/mL progastrin (original magnification, ×40).
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12. Supplementary Figure 4
Progastrin specifically binds to CCK2R. Immunofluorescence of progastrin binding to AGSE cells but not to AGS cells (white arrows) during co-culture (original magnification, ×300). (B) Immunofluorescence analysis of progastrin binding to AGSE cells with or without YF476 pretreatment (original magnification, ×600). DAPI, 4′,6-diamidino-2-phenylindole.
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13. Supplementary Figure 5
Progastrin binding to AGSE cells increases over time. Immunofluorescence for progastrin binding on AGSE and AGS (white arrows) cells at different time points (original magnification, ×300). DAPI, 4′,6-diamidino-2-phenylindole.
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14. Supplementary Figure 6
Progastrin binding to CCK2R is temperature sensitive. Immunofluorescence for progastrin binding on Colo320(+) and Colo320(−) cells at 4°C (original magnification, ×300). DAPI, 4′,6-diamidino-2-phenylindole.
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15. Supplementary Figure 7
Progastrin treatment leads to CCK2R dependent proliferation in gastric cancer cells. MTT assay of AGSE (A) and AGS (B) cells after progastrin treatment (n = 6/group). Quantitative RT-PCR analysis of Bmp2 mRNA expression levels after progastrin treatment in AGSE (C) and AGS (D) cells (n = 3 plates/group). Expression levels were normalized to reference gene glyceraldehyde-3-phosphate dehydrogenase. All values represent the mean ± SD. ∗∗P < .01.
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16. Supplementary Figure 8
Quantitative RT-PCR analysis of β-arrestin 1 (A) and 2 (B) mRNA levels in Colo320 after β-arrestin 1 and 2 knockdown through siRNA (50 ng, 200 ng/140 uL) (n = 3 plates/group). qRT-PCR analysis of Bmp2 mRNA expression levels after treatment with CCK2R antagonist, YF476 to the Colo320(+) (C) and Colo320(−) (D) cells (n = 3 plates/group). Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase. (E) Bmp2 promoter luciferase assay of Colo320(+) and Colo320(−) cells after progastrin treatment (n = 3 plates/group). All values represent the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
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17. Supplementary Figure 9
Progastrin increases colonic stem cell numbers. (A) Representative pictures of Lgr5-GFP and hGAS/LGR5-GFP mice colonic crypts. (B) Quantification of Lgr5-GFP−positive cells in Lgr5-GFP and hGAS/Lgr5-GFP mice (n = 6 colons/group). All values represent the mean ± SD. ∗∗P < .01.
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18. Supplementary Figure 10
Progastrin leads to increased symmetric cells divisions. (A) Representative pictures of immunofluorescence for Numb-positive Colo320(+) cells after symmetric and asymmetric cells divisions (original magnification, ×600). (B) Fluorescence-activated cell sorting analysis for Numb-positive cells in Colo320(+) and Colo320(−) cells after progastrin treatment. Horizontal axis: Numb-phycoerythrin (PE). (C) Percentage of Numb-positive cells after progastrin treatment to the AGSE and AGS cells (n = 3 plates/group). All values represent the mean ± SD. ∗∗P < .01. DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast.
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