Fluorescein isothiocyanate (fitc)

For the intake ability test, 2 × 10⁴ cells were seeded in 96 black wells for 1 day and treated with 100 μM FITC (Millipore-Sigma) for 5, 10, 30, and 60 min. Subsequently, the cells were washed with 1× PBS thrice and detected using the Fluorescent Cell Imager (ZOE; Bio-Rad, Hercules, CA, USA).
For the declining test, 2 × 10⁴ cells were seeded in 96 black wells for 1 day and treated with 100 μM FITC for 60 min. Subsequently, the cells were washed with 1x PBS thrice and detected using the Fluorescent Cell Imager (ZOE; Bio-Rad, Hercules, CA, USA) at 0, 0.5, 1, and 3 h after FITC treatment.
All images were further analyzed using ImageJ for quantification. After acquiring the pixel number from GFP, the pixel number of the untreated sample was excluded from all data to reduce the noise from the background.

Since antibodies to the oestrogen receptor were raised in the same species, Anti‐ERα (Saint John's Laboratory, UK) and Anti‐GPER1 (Saint John's Laboratory, UK) were conjugated to fluorescein isothiocyanate (FITC) (Thermofisher, UK), as per the manufacturers’ protocols (Thermofisher UK). Briefly, Anti‐ERα and Anti‐GPER1 were initially concentrated using Amicon Ultra centrifugal filter units (Ultra 4 MWCO 30KDA) (Merck Millipore, UK) to generate stock solutions of concentrated Anti‐ERα and Anti‐GPER1 at 2 mg/ml in 0.1 M sodium bicarbonate. FITC stock solution was prepared at 1 mg/ml in anhydrous dimethyl sulphoxide (DMSO). Then, 50 μl of FITC solution was slowly add to the 2 mg/ml of antibody solution and incubated at 4°C for 1 h with continuous stirring. Free fluorophores were separated from conjugated antibody using PD‐10 column, Sephadex G‐25 M (BioRad, UK) and conjugated ERα/ FITC and GPER/FITC was eluted with PBS. In order to determine the labelling efficiency and concentration, absorbance values were measured at A280 and A495. For effective labelling, the degree of labelling should fall within 2–6 mol of FITC per 1 mol of antibody; typical concentrations were in the range of 0.2 mg/ml. The newly conjugated antibody was stored at −20°C until use.