Ab92377

MCF-7 cells were cultured in DMEM with 5% charcoal-dextran stripped FBS for 1 day to eliminate any estrogenic source before treatment, then the cells were treated with QYFE (0.00001~0.01mg/mL), 17β-estradiol (0.01 μM) with or without 0.1 μM ICI182, 780, 0.1% DMSO treatment as negative control. After 48h, all cells were harvested protein. Rabbit anti-ER α monoclonal antibody (dilution 1/1000, ab32063, Abcam Biotechnology, UK), rabbit anti-ERβ polyclonal antibody (dilution 1/2000, ab3577, Abcam Biotechnology, UK), rabbit anti-pS2 monoclonal antibody (dilution 1/1000, ab92377, Abcam Biotechnology, UK), rabbit anti-PR monoclonal antibody (dilution 1/1000, ab133526, Abcam Biotechnology, UK) were used, and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (dilution 1/5000, ab181602, Abcam Biotechnology, UK) was used as internalcontrol. Finally incubated with goat anti-Rabbit IgG polyclonal antibodyHRP-Labelled (1:4000; C1309; Beijing Pulilai Gene Technology Co., Ltd.). All determinations were carried out in triplicate. Visualization of protein bands was accomplished using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) [39 (link)].

After RIPA protein extraction reagent (P0013; Beyotime; Beijing, China) supplied with phenylmethylsulfonyl fluoride and protease inhibitor treatment, extracted proteins (20 μg) from transfected cells were determined as for its protein density and went through separation with 10% SDS‐PAGE, followed by immediate transfer onto a PVDF membrane (Cat. no: 3010040001; Roche; Shanghai, China). Separated proteins went through an overnight incubation with anti‐Bcl‐2 (1:1000 dilution; ab182858; abcam), anti‐Bax (1:1000 dilution; ab182733; abcam), anti‐cleaved caspase‐3 (1:1000 dilution; ab2302; abcam), anti‐caspase‐3 (1:1000 dilution; ab13585; abcam), anti‐E‐cadherin (1:1000 dilution; ab1416; abcam), anti‐N‐cadherin (1:1000 dilution; ab18203; abcam), anti‐MMP2 (1:1000 dilution; ab37150; abcam), anti‐MMP9 (1:1000 dilution; ab38898; abcam), anti‐TFF1 (1:1000 dilution; ab92377; abcam), anti‐GADPH (1:1000 dilution; ab8245; abcam) at 4°C and 1 hour incubation with Rabbit Anti‐Mouse IgG H&L (HRP) (1:2000 dilution; ab6728; abcam) in 5% non‐fat milk at indicated dilution. Densitometer (Cat. no: GS800; Quantity One software; Bio‐Rad, Hercules, CA, USA) detected the band intensities with GADPH as reference gene.

Formalin-fixed paraffin-embedded tissues were cut int 4 μm sections which were deparaffinized with xylene and rehydrated through a graded series of alcohols. The tissue sections were placed in a repair box filled with citric acid antigen retrieval solution, and antigen retrieval was performed in a microwave oven. The slides were washed 3 times with PBS after natural cooling, 5 min each time and then, blocking was done with 3% BSA in PBS buffer for 30 min. Tissues were incubated with primary antibodies against Rev-erbα (ab174309, Abcam) and Tff1 (ab92377, Abcam) at 1 : 200 dilutions overnight at 4°C in humidified chambers followed by incubation with the corresponding secondary antibody for 60 min at room temperature in the dark. The slides were mounted with resin after dropping an appropriate amount of an antifluorescence quencher on the tissue. Fluorescence was detected with fluorescence microscopy. Fluorescence was detected by using a LEICA TCS NT laser confocal microscope.