Phenol red

To test the effects on a variety of different breast cancer cells, we purchased human ERα-positive (MCF-7, T47D) and ERα-negative (HCC1937, SW527) breast cancer cell lines as well as immortalized breast epithelial cells (MCF10A and 184B5) from ATCC. All cells were maintained as monolayer cultures. MCF7 and T47D were cultured in MEM media supplemented with 10% FBS and 0.01 mg/ml insulin (Hyclone, Utah, USA). MCF10A and 184B5 cell lines were cultured in DMEM-F12 media supplemented with 2 mM L-glutamin, 1% Penicillin/Streptomycin, 200 ng/ml EGF, 100 ng/ml Cholera toxin, 0.01 mg/ml insulin, 500 ng/ml Hydrocortisone, 5% Horse serum (Gibco, California, USA). HCC1937 was cultured in RPMI-1640 with 10% FBS. SW527 was cultured in DMEM media supplemented with 10% FBS. If necessary, MCF7 cells were cultured in MEM free of phenol red (Gibco) and T47D cells were cultured in RPMI-1640 free of phenol red with charcoal-stripped serum (Biological Industries, Kibbutz BeitHaemek, Israel).

For live cell imaging, 35 mm glass-bottomed dishes (MatTek Corporation, Ashland, MA, United States) were coated with 10 μg/ml fibronectin (#F2006, Sigma Corp., St. Louis, MO, United States) in PBS for at least 3 h at 37°C, washed with PBS twice and immersed in complete DMEM medium without phenol red (#01-053-1A, Biological Industries, Kibbutz Beit-Haemek, Israel) before seeding of cells. The time-lapse images of cells with transient transfection of CAV-1-mEGFP and mCherry-actin were acquired with Olympus cellSens Dimension system, consisting of an Olympus SpinSR10 Ixplore spinning disk confocal and a Yokogawa CSU-W1 confocal scanner. Appropriate filters, heated sample environment (+37°C), controlled 5% CO₂ and UplanApo 100×/1.5 Oil objective (Olympus Corporation, Tokyo, Japan) was used. The recording was set as every 1 s for 200 s and one focal plane was recorded for all live cell videos. For tracking and speed measurement of CAV-1 vesicles, the Imaris 9.2 (Bitplane, Zurich, Switzerland) “Track” module with globular-objects over time was used as in previous study (Jiu, 2018 (link)). Two micrometers estimated XY diameter, 5 μm max distance and 3 max gap size were set for analyzing.

The ER+ breast cancer cell lines MCF7, T47D and CAMA1 and the triple-negative breast cancer cell lines MDAMB231, MDAMB468 and HCC1143 were cultured in RPMI-1640 medium with phenol red (Biological Industries), supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 mg/mL penicillin (Gibco) and 100 mg/mL streptomycin (Gibco). All the cell lines were cultured at 37° C in a humidified atmosphere with 5% (v/v) CO₂ in the air. The MCF7, T47D and MDA-MB-231 cell lines were kindly provided by Dr. Nuria Vilaboa (La Paz University Hospital, previously obtained from ATCC in January 2014). The MDAMB468, CAMA1 and HCC1143 cell lines were obtained from ATCC (July 2014). Cell lines were routinely monitored in our laboratory and authenticated by morphology and growth characteristics, tested for Mycoplasma and frozen, and passaged for fewer than 6 months before experiments. The MTF (Sigma Aldrich D150959) and RP (Sigma Aldrich R8781) were obtained from Sigma-Aldrich (St. Louis, MO, USA).