1. Volume 139, Issue 6, Pages 2113-2123 (December 2010)
NOTCH1 and NOTCH3 Coordinate Esophageal Squamous Differentiation Through a CSL-Dependent Transcriptional Network 
Shinya Ohashi, Mitsuteru Natsuizaka, Yumi Yashiro–Ohtani, Ross A. Kalman, Momo Nakagawa, Lizi Wu, Andres J. Klein–Szanto, Meenhard Herlyn, J. Alan Diehl, Jonathan P. Katz, Warren S. Pear, John T. Seykora, Hiroshi Nakagawa 
Gastroenterology 
Volume 139, Issue 6, Pages (December 2010)
DOI: /j.gastro
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2. Figure 1
Ca2+ activates Notch signaling to induce squamous differentiation in esophageal keratinocytes. EPC2-hTERT cells were exposed to Ca2+ at indicated concentrations in the presence or absence of GSI or DNMAML1 for (A and C) 48 hours after transfection or (B and D–F) 72 hours. (A) 8xCSL-luc reporter activities, (B) HES5 mRNA levels, (C) IVL-luc reporter activities, (D) IVL mRNA levels, (E) IVL protein levels, and (F) DNMAML1 and IVL protein levels. Luciferase assays, real-time RT-PCR, and Western blotting determined reporter activities, mRNA, and protein, respectively. β-actin served as an internal or loading control in B, D, E, and F. GSI (−), dimethyl sulfoxide only; GSI (+), 1 μmol/L compound E; GFP, control vector in A–D and F. *P < .001 vs 0.09 mmol/L Ca2+ + GSI (−) or GFP; #P < .001 vs 0.6 mmol/L Ca2+ + GSI (−) or GFP; n = 6 in A and C; n = 3 in B and D.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Notch inhibition impairs squamous differentiation of esophageal epithelia reconstituted in organotypic 3D culture. EPC2-hTERT cells were grown in (A–C and E) 3D and (D) monolayer cultures in the presence or absence of 1 μmol/L compound E (GSI) or DNMAML1 and subjected to (A and E) H&E staining, (B) immunohistochemistry for Ki67, and (C) immunofluorescence for CK14 (red) and IVL (green). Ki67 labeling index in B was 36.7 ± 9.7 for dimethyl sulfoxide, 32.8 ± 4.2 for GSI, 33.9 ± 5.4 for GFP, and 36.8 ± 3.8 for DNMAML1 without significant difference (n = 6). In D, Western blotting determined DNMAML1 repression by doxycycline (DOX) at indicated concentrations in the Tet-Off system. DOX (−), 0 μg/mL DOX; DOX (+), 2 μg/mL DOX in E. Scale bar = 50 μm. Ep., epithelium; Str., stroma.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
N1 and N3 expression in normal human esophageal epithelium. Representative H&E and immunohistochemistry. N1 and N3 were detected in the nucleus (arrows) and on the membrane (arrowheads). Scale bar = 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Sequential induction and activation of N1 and N3 during Ca2+-induced squamous differentiation in esophageal keratinocytes. EPC2-hTERT cells were exposed to 0.6 mmol/L Ca2+ (A–C) for indicated time periods or (D and E) 72 hours and subjected to (A and E) real-time RT-PCR or (B–D) Western blotting. In D and E, Ca2+ stimulation was performed in the presence or absence of GSI or DNMAML1. Compound E (GSI) was used at indicated concentrations. β-actin served as an internal or loading control in A, B, D, and E. Histone H1 served as a loading control for nuclear extracts in C. (A) *P < .001 vs time 0 (n = 3). (E) *P < .001 vs 0.09 mmol/L Ca μmol/L GSI or GFP; #P < .001 vs 0.6 mmol/L Ca μmol/L GSI or GFP (n = 3). Arrowheads indicate FL-N3 and brackets indicate doublets of ICN3.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
ICN1 directly induces and activates N3 through CSL-dependent transcription in esophageal keratinocytes. EPC2-hTERT cells expressing either DNMAML1 or GFP (control) were transduced with ICN1 to determine (A) N3 mRNA and (B) N3 protein levels 72 hours after retrovirus infection. In C and D, EPC2-hTERT cells were stimulated with Ca2+ for 72 hours following N1 small interfering RNA transfection to determine indicated molecules. Real-time RT-PCR and Western blotting determined mRNA and protein, respectively. β-actin served as an internal or loading control in A–D. *P < .001 vs GFP + control (empty MigRI vector) or 0.09 mmol/L Ca2+ + small interfering RNA control; #P < .001 vs ICN1 + GFP or 0.6 mmol/L Ca2+ + small interfering RNA control; n = 3 in A and C. Closed arrowheads indicate FL-N3, open arrowheads indicate ICN3, and brackets indicate doublets of ICN3. In E, cells were stimulated with Ca2+ for 48 hours for ChIP assays with indicated antibodies. *P < .01, **P < .05 vs 0.09 mmol/L Ca2+; n = 3.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

7. Figure 6
N3 is required and cooperates with N1 during esophageal squamous differentiation. EPC2-hTERT cells stably expressing 2 independent short hairpin RNA sequences directed against N3 (N3-A and N3-B) or control short hairpin RNA were stimulated with 0.6 mmol/L Ca2+ for (A, B, and D) 72 hours or (C) 48 hours after IVL-luc transfection. (A), N3 mRNA; (B), ICN1, ICN3, and IVL protein levels; (C), IVL-luc activities; and (D), IVL mRNA levels. Luciferase assays, real-time RT-PCR, and Western blotting determined reporter activities, mRNA, and protein, respectively. β-actin served as an internal or loading control. *P < .001 vs 0.09 mmol/l Ca2+ + scramble; #P < .001 vs 0.6 mmol/L Ca2+ + scramble. n = 3 in A and D; n = 6 in C. In E, cells were grown in organotypic 3D culture and subjected to H&E staining (top panels), immunohistochemistry for Ki67 (middle panels), and immunofluorescence for CK14 (red) and IVL (green) (lower panels). Scale bar = 50 μm. Ep., epithelium; Str., stroma. In F, EPC2-hTERT cells were transfected with IVL-luc along with an empty vector (200 ng), ICN1 (200 ng), ICN3 (200 ng), or in combination (100 ng each) for luciferase assays. *P < .001 vs empty; ns, not significant vs empty. n = 6 in F.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
DNMAML1 induce basal cell hyperplasia/dysplasia impairing squamous differentiation and N3 expression in the mouse esophageal epithelium. Representative K14Cre;DNMAML1 and control (DNMAML1f/f and K14Cre) mouse esophagi were subjected to H&E staining; immunohistochemistry for Ki67, Flg, and N3; or immunofluorescence for Ivl. Ki67 labeling index, 19.9% ± 5.0% for DNMAML1f/f, 21.8% ± 4.8% for K14Cre (not significant vs DNMAML1f/f); and 69.6% ± 5.5% for K14Cre;DNMAML1 (P < .001 vs DNMAML1f/f and K14Cre) (n = 6). Arrows denotes Ivl cytoplasmic staining, Flg localization to keratohyalin granules, and nuclear N3 staining in each panel. Scale bar = 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
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