Figure 3

NOTCH1 regulates keratin expression. (a) Expression of NOTCH1, NOTCH2 and NOTCH3 in cervical cancer cell lines (HeLa, CaSki), oral cancer cell lines (BHY, Ca9-22, HSC-3) and primary human foreskin (HFS) cells, as revealed by western blot. All the antibodies recognize an epitope in the intracellular domain. Most proteins were detected as a furin-cleaved form containing the transmembrane and intracellular domains. A small amount of full-length protein was also detected. Gamma-secretase-cleaved C-terminal fragment (NICD) was not observed in NOTCH1 and NOTCH3 blots. (b) Expression of various keratins (K), vimentin (Vim), E-cadherin (Cdh1), desmoglein 3 (Dsg3) and TP63 in various cells, as revealed by western blot. As the full-length NOTCH1 was observed at a much weaker intensity and in proportion to the intensity of the 120 kDa protein, only the 120 kDa protein of NOTCH1 is shown. (c) K13 expression in Ca9-22 and HFS cells cultured at a high density. Immunofluorostaining was conducted using the secondary antibody labeled by Alexafluor 488. The nuclei were counterstained by DAPI. K13-expressing cells appear in a scattered manner. HeLa, CaSki, BHY and HSC-3 showed no K13 expression. (d) Ca9-22 cells were treated with only DMSO or 2.5 μM DAPT at 80% confluency and were cultured for 72 h. An equal amount of protein was subjected to western blot analysis for keratins (K), HES1, HEY1 and β-tublin (βTub). (e) Ca9-22 cells were transfected at 30% confluency with an empty plasmid (Ctrl), NICD, dominant-negative RBPJ (R218H, RBPJm), dominant-negative Dll1 (dnDl) or siRNA for NOTCH1 (siN1) and were cultured for 72 h. (f) Ca9-22 cells were transfected with TP63(TA) or TP63(ΔN) and were incubated for 72 h. Western blot analysis revealed the endogenous expression of TP63 (ΔN), but not TP63 (TA). NOTCH1 was only slightly downregulated by TP63 (ΔN). K13 was downregulated both by TP63 (TA) and TP63 (ΔN). (g) Ca9-22 cells were transfected with NICD or siRNA for NOTCH1 and were incubated for 72 h. Both NICD and siN1 downregulated TP63 and K13. (h) Luciferase reporter assay for human KRT13, KRT15 and KRT17 promoters. The promoter construct plus of 0.1 μg and 0.1 μg of the Notch1 construct and 0.01 μg of the Renilla luciferase standardization plasmid (pEF-RL). Transfections were done into GE-1 cells on 48-well plates and the luciferase activity was measured 48 h after transfection using Dual-Luciferase Reporter Assay System (Promega). The error bars denote standard errors. rlu; relative luciferase unit.