EGF promotes EMT but does not induce RIP of EpCAM.

(A) FaDu and Kyse30 cells were treated with control media and a low (1.8 nM) and high (9 nM) dose of EGF. Cell morphology was assessed after 72 hr. Shown are representative micrographs at 100× magnification from 3 independent experiments. (B) FaDu and Kyse30 cells were treated with control media and a high (9 nM) dose of EGF. Expression of E-cadherin was assessed by immunofluorescence staining and confocal laser scanning microscopy after 72 hr. Shown are representative staining from 3 independent experiments. (C) FaDu and Kyse30 cells were treated with control media (“ctrl.”) and high (9 nM) dose of EGF. Expression of E-cadherin was assessed by IB after 72 hr. Shown are representative immunoblots (upper panels) and mean values with SDs from n = 3 independent experiments. ** p-value p-value t test. Supporting data are compiled in S1 Data. (D) Schematic representation of EpCAM RIP including proteases involved and inhibitors. (E) Immunoprecipitation of the EpEX from supernatants of Kyse30 and HCT8 cells expressing EpCAM-YFP with or without EGF 9 nM. Shown are representative results from n = 3 independent experiments. (F) Visualization of CTF-EpCAM-YFP in membrane isolates of Kyse30 and HCT8 cells expressing EpCAM-YFP with or without EGF 9 nM. Shown are representative results from n = 3 independent experiments. (G) Visualization of EpCAM-YFP, CTF-YFP, and EpICD-YFP in Kyse30 and HCT8 and Kyse30 cells expressing EpCAM-YFP with or without EGF 9 nM in the presence of proteasome inhibitor ß-lactone (“ß-lac,” 50 μM). Shown are representative results from n = 3 independent experiments. (H) Visualization of EpICD-YFP in cytoplasmic (“cyt.”) and nuclear fractions (“nuc.”) of Kyse30 and HCT8 cells expressing EpCAM-YFP with or without EGF 9 nM in the presence of proteasome inhibitor (“ß-lac,” 50 μM). Tubulin and lamin A staining served to control fractionated samples. Shown are representative results from n = 3 independent experiments. (I, J) Indicated cell lines were treated with EGF 1.8 nM and 18 nM for 24 hr, 9 nM for 72 hr, or 1.8 nM TGFα for 24 hr. Expression of EpCAM in control (“w/o EGF”) and treated cells was assessed by flow cytometry (I) and IB (J). Shown are ratios of EpCAM mean fluorescence intensities divided by control intensities (I; “EpCAM/iso”) and relative fold changes of EpCAM expression in whole-cell lysates (J; relative EpCAM fold change), where expression levels in control cells were set to 1 for comparison. Shown are means with SDs from n = 3 independent experiments. ** p-value t test. Supporting data are compiled in S1 Data. (K) Indicated cell lines were treated with EGF 9 nM for 72 hr, and cell surface expression of EpCAM was assessed by fluorescence immunostaining and laser scanning confocal microscopy. EpCAM: green, nuclei: blue (DAPI). Shown are representative results from n = 3 independent experiments with multiple areas analyzed. CTF, C-terminal fragment; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; EGF, epidermal growth factor; EMT, epithelial-mesenchymal transition; EpCAM, epithelial cell adhesion molecule; EpEX, extracellular domain of EpCAM; IB, immunoblotting; MW, molecular mass; RIP, regulated intramembrane proteolysis; SD, standard deviation; TGFα, transforming growth factor alpha; YFP, yellow fluorescent protein.