1. Volume 139, Issue 3, Pages 918-928.e6 (September 2010)
Atonal Homolog 1 Is Required for Growth and Differentiation Effects of Notch/γ- Secretase Inhibitors on Normal and Cancerous Intestinal Epithelial Cells 
Avedis Kazanjian, Taeko Noah, Douglas Brown, Jarred Burkart, Noah F. Shroyer 
Gastroenterology 
Volume 139, Issue 3, Pages e6 (September 2010)
DOI: /j.gastro
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2. Figure 1
Atoh1 mediates GSI differentiation into secretory cells. (A) H&E staining of distal ileum. Vehicle- and GSI-treated (GSI-20, 10 μmol/L/kg twice a day for 5 days) WT and Atoh1Δintestine mice. (B) Alcian blue/periodic acid–Schiff staining of ileal sections. Arrows point to goblet cells in the villi and crypts. (C) Lysozyme staining of Paneth cells. Middle panels represent magnification of ileal crypts of the smaller highlighted boxes. (D) Chromogranin staining of the ileal tissues. Middle panels represent magnification of the smaller highlighted boxes; arrows point to chromogranin-positive cells.
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3. Figure 2
Atoh1 mediates GSI-induced secretory cell gene expression in the intestine. Quantitative RT-PCR for gene expression in ileal and colonic tissues treated with vehicle or GSI (10 μmol/L/kg once a day for 5 days), normalized to glyceraldehyde-3-phosphate dehydrogenase expression. White bars represent WT tissues and black bars represent Atoh1Δintestine tissues. P values shown were calculated using 2-way ANOVA for drug (GSI) and genotype (WT vs Atoh1Δintestine) effects. *P < .05, **P < .001, ***P < .0001, ****P < The error bars represent the standard error of the mean.
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4. Figure 3
ATOH1 mediates GSI-induced secretory cell gene expression in colon cancer cells. (A) Alcian blue/periodic acid–Schiff staining of LS174T cells treated either with DMSO or GSI (5 μmol/L DAPT) for 4 days. The insets are higher magnification of stained cells. (B) Quantitative RT-PCR for gene expression in LS174T cells treated with DMSO or GSI (5 μmol/L DAPT), normalized to expression at day 0. P values represent GSI effect using 2-way ANOVA. (C) Quantitative RT-PCR for gene expression in LS174T cells transduced with lentivirus encoding either nontargeting (white bars), or ATOH1-targeting (grey and black bars) shRNA, normalized to expression of cells transduced with nontargeting shRNA at day 0. Forty-eight hours after transduction (day 0) cells were treated with DMSO or GSI (5 μmol/L DAPT) for 4 days. P values shown were calculated using 2-way ANOVA for shRNA effect; Bonferroni posttests calculated individual group differences, *P < .05, **P < The error bars represent the standard error of the mean. (D) A representative immunoblot of LS174T after 4 days of treatment with DMSO or GSI (5 μmol/L DAPT). Antibodies recognized the Notch intracellular domain (NICD), HES1, trefoil factor 3 (TFF3), and actin (as a loading control).
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5. Figure 4
Atoh1 mediates GSI-induced growth inhibition in normal intestinal epithelia. (A) S-phase cells (marked by BrdU) were stained brown in ileal sections from WT and Atoh1Δintestine mice treated with vehicle or GSI (GSI-20, 10 μmol/L/kg twice a day for 5 days). White lines underline WT crypts from WT animals; grey lines underline Atoh1-WT crypts in Atoh1Δintestine mice, and black lines underline Atoh1-null crypts in Atoh1Δintestine mice. (B) Quantification of BrdU-positive ileal cells from vehicle- or GSI-treated WT and Atoh1Δintestine mice. Animals were treated for 5 days with 10 μmol/L/kg once a day (GSI) or twice a day (2 × GSI). The white, grey, and black bars represent counts from different sets of crypts as described in panel A. (WT animals: n = 5 for vehicle, n = 4 for GSI, and n = 7 for 2 × GSI; Atoh1Δintestine animals: n = 4 for vehicle, n = 8 for GSI, and n = 5 for 2 × GSI.) A significant difference in proliferation was measured for vehicle-treated WT vs Atoh1-null crypts (P < .01), and Atoh1-WT vs Atoh1-null crypts (P < .001). (C) Colonic sections from mice as shown in panel A. (D) Quantification of BrdU-positive colonic cells from mice as shown in panel B. (WT animals: n = 3 for vehicle, n = 8 for GSI, and n = 6 for 2 × GSI; Atoh1Δintestine animals: n = 4 for vehicle, n = 4 for GSI, and n = 5 for 2 × GSI.) (B and D) Error bars represent the standard error of the mean. Significance was calculated using 2-way ANOVA for GSI and genotype effects. Bonferroni posttest analysis was used for calculating significance for individual groups. *P < .05, **P < .001.
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6. Figure 5
GSI-induced ATOH1 expression level is associated with CRC growth inhibition. (A) CRC cell lines are named above immunoblots and pairs of bar graphs representing cell growth (upper bar graph) and BrdU incorporation (lower bar graph) after 4 days of treatment with DMSO or GSI (5 μmol/L DAPT). Antibodies recognized the Notch intracellular domain (NICD), and actin (as a loading control) for the representative immunoblots. Cell growth and proliferation were quantified by cell viability and BrdU incorporation assays and normalized to growth or BrdU incorporation of DMSO-treated cells. (B) Log-linear comparison of the GSI effect on cell growth vs ATOH1 expression (determined by quantitative RT-PCR, Table 1) for CRC cell lines. Linear regression was used to determine the model shown and to determine R2. GSI-induced ATOH1 expression represents the ratio of DMSO/DAPT for normalized expression levels of ATOH1. The GSI-induced growth arrest represents the ratio of DMSO/DAPT for the normalized average cell count for each treatment. Error bars represent the standard error of the mean. Significance was calculated using 2-tailed Student t tests comparing DMSO with GSI treatment. *P < .05, **P < .001.
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7. Figure 6
Atoh1 mediates GSI-induced differentiation and growth inhibition in intestinal tumors. (A) Quantification of proliferation in ileal adenomas from mice treated with vehicle or GSI (GSI-20, 10 μmol/L/kg twice a day for 5 days). White bars represent adenomas from APCmin animals. Grey bars represent Atoh1-WT adenomas in APCmin;Atoh1Δintestine mice. Black bars represent Atoh1-null adenomas from APCmin;Atoh1Δintestine mice. (APCmin adenomas: n = 21 for vehicle, n = 33 for GSI; APCmin;Atoh1-WT adenomas: n = 27 for vehicle, n = 25 for GSI; APCmin;Atoh1-null adenomas: n = 15 for vehicle, n = 14 for GSI.) A significant difference in proliferation was measured for vehicle-treated WT vs Atoh1-null adenomas (P < .001), and Atoh1-WT vs Atoh1-null adenomas (P < .001). (B) Quantification of proliferation in colonic adenomas from mice treated with vehicle or GSI (GSI-20, 10 μmol/L/kg twice a day for 5 days). The color scheme for bars is the same as in panel A. (APCmin adenomas: n = 9 for vehicle, n = 16 for GSI; APCmin;Atoh1-null adenomas: n = 10 for vehicle, n = 19 for GSI.) Error bars represent standard error of the mean. Significance was calculated using 2-way ANOVA for GSI and genotype effects. Bonferroni posttest analysis was used for calculating significance for individual groups. *P < .05, **P < (C and D) Representative alcian blue/periodic acid–Schiff staining of APCmin and APCmin;Atoh1-null ileal and colonic adenomas treated with vehicle and GSI (GSI-20, 10 μmol/L/kg twice a day for 5 days).
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8. Supplementary Figure 1
GSI-20 and DAPT inhibit Notch signaling in LS174T cells. Representative immunoblots of LS174T cell lysates treated with DMSO or DAPT (5 μmol/L) or GSI-20 (0.5 μmol/L) for 4 days. Antibodies recognized HES1 and actin (as a loading control).
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9. Supplementary Figure 2
GSI-20 inhibits Notch signaling in the colon. Representative immunoblots of colonic lysates from WT and Atoh1Δintestine animals treated with vehicle or GSI (GSI-20, 10 μmol/L/kg twice a day for 5 days). Antibodies recognized the Notch intracellular domain (NICD), p27, Tff3, and actin (as a loading control).
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10. Supplementary Figure 3
Atoh1 and GSI regulated apoptosis in normal and adenomatous intestinal epithelium. (A and B) Quantification of immunohistochemical cleaved caspase-3 staining in vehicle or GSI-treated (GSI-20, 10 μmol/L/kg twice a day for 5 days) (A) ileal and (B) colonic crypt sections. White bars represent WT crypts from WT animals; grey represents Atoh1-WT crypts in Atoh1Δintestine mice, and black represents Atoh1-null crypts in Atoh1Δintestine mice. (WT animals: n = 5 for vehicle, n = 7 for GSI; Atoh1Δintestine animals: n = 5 for vehicle, n = 5 for GSI.) Significance was calculated using 2-way ANOVA for drug (P < for both ileum and colon) and genotype (P < for ileum) effects. Bonferroni posttest analysis was used for calculating significance for individual groups. **P < .05, **P < (C and D) Quantification of cleaved caspase-3 staining of vehicle and GSI-treated (GSI-20, 10 μmol/L/kg twice a day for 5 days) ileal and colonic adenomas. (Ileal adenomas: APCmin adenomas: n = 31 for vehicle, n = 37 for GSI; APCmin;Atoh1-WT adenomas: n = 28 for vehicle, n = 44 for GSI; APCmin;Atoh1-null adenomas: n = 19 for vehicle, n = 14 for GSI. Colonic adenomas: APCmin adenomas: n = 9 for vehicle, n = 18 for GSI; APCmin;Atoh1-null adenomas: n = 7 for vehicle, n = 27 for GSI.) Significance was calculated using 2-way ANOVA for drug (P > .2 for both ileal and colon adenomas) and genotype (P < .02 for both ileal and colon adenomas) effects. Error bars represent the standard error of the mean for all graphs.
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11. Supplementary Figure 4
GSI treatment of CRC cell lines does not induce apoptosis. Representative immunoblots of CRC cells after 4 days of treatment with DMSO or GSI (5 μmol/L DAPT). Control lane contains lysate from ultraviolet-treated LS174T cells. Antibodies against poly-ADP ribose polymerase (PARP) recognize full-length and the major cleavage product induced by apoptosis (C-PARP). Actin was used as a loading control.
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12. Supplementary Figure 5
H&E-stained sections of APCmin and APCmin;Atoh1Δintestine adenomas. H&E stain of (A) ileal and (B) colonic adenomas from vehicle- and GSI-treated (GSI-20, 10 μmol/L/kg twice a day for 5 days) APCmin and APCmin;Atoh1Δintestine mice. The panels to the left show APCmin adenomas and the panels to the right show APCmin;Atoh1-null adenomas.
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13. Supplementary Figure 6
Atoh1 mediates GSI-induced reduction of proliferation in colonic tumors. Representative immunohistochemical staining for BrdU in colonic adenomas from vehicle- and GSI-treated (GSI-20, 10 μmol/L/kg twice a day for 5 days) APCmin and APCmin;Atoh1Δintestine mice. The panels on the left show APCmin adenomas and the panels on the right show APCmin;Atoh1-null adenomas at 40× magnification.
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