Eea1 ab70521

Cell lines HeLa, HepG2, SK-Hep-1, RKO, U87, Huh7 and 293T were purchased from the Chinses Academy of Science and grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). All cell lines were incubated at 37 °C in a humidified 5% CO₂ atmosphere. Antibodies including NUMB (2756S), N-cadherin (13116S), E-cadherin (3195S), Vimentin (5741S), Fibronectin (26836S), pan-keratin (4545T), Twist1 (69366S), SMAD3 (9523S), p-SMAD3 (9520T) and Slug (9585S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies such as β-actin (sc-47778), α-Tubulin (sc-73242) and Notch1 (sc-376403) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies EEA1 (ab70521) and LAMP1 (ab25631) were from Abcam, and Notch1 (AF1546), Ubiquitin (AF1705) and Histone3 (AF0009) from Byotime (Nantong, China).

Mammary gland cryosections were processed as described above for immunohistochemistry and evaluated by confocal microscopy. HMEpCs were plated in 8-well chamber slides (Nunc, Thermo Fisher Scientific) at 1 × 10⁴ cells per well and were allowed to adhere overnight. Cells were washed with PBS, fixed for 15 min with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 10 min. For both tissues and cells, nonspecific sites were blocked for 1 h at room with 3% BSA, washed in PBS-T and incubated at 4 °C overnight with primary antibodies: Notch 1 (antibody clone EP1238Y, ab52627), EEA1 (ab70521), TGF-β1 (ab66043), pSmad2 (ab188334) and Ki67 (ab15580) were from Abcam, Cambridge, MA, USA; cytokeratin 18 (Fitzgerald, North Acton, MA, USA; 70R-30585) and cytokeratin 14 (Thermo Fisher Scientific; MA5-11599). After primary antibody incubation, slides were washed three times with PBS and incubated with secondary antibodies: Alexa Fluor 488 goat anti-rabbit (A-11008) and Alexa Fluor 594 donkey anti-mouse (A-21203) (Invitrogen, Thermo Fisher Scientific) for 1 h at room temperature. The stained slides were imaged on an Olympus Fluoview Fv10i confocal microscope (Olympus, Waltham, MA, USA). Analysis was performed using Fv10i Flouview Ver.3.0 software (Olympus).