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 48 hrs after transfection in (A) and (C), or 72 hrs in (B), (D)–(F). (A) 8×CSL-luc reporter activities, (B) HES5 mRNA levels, (C) IVL-luc reporter activities, (D) IVL mRNA levels, (E) IVL protein levels, (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 (−), DMSO only; GSI (+), 1 μM compound E; GFP, control vector in (A)–(D) and (F). *, P< 0.001 vs. 0.09 mM Ca2+ + GSI (−) or GFP; #, P< 0.001 vs. 0.6 mM Ca2+ + GSI (−) or GFP; n=6 in (A) and (C); n=3 in (B) and (D).