Quantifying EGF Internalization in HTR-8/SVneo Cells

EGF internalization was evaluated in HTR-8/SVneo cells using a modified EGF endocytosis assay [24 (link)]. After overnight serum starvation, cells were pre-treated with BPS (1 or 10 μg/mL) in 0.1% DMSO for 5 min, followed by a 5 min co-exposure with BPS (1 and 10 µg/mL) and Alexa Fluor Texas Red-conjugated EGF (100 ng/mL; biotinylated EGF complexed to Alexa Fluor 647 Streptavidin, Cat#: E13345, Thermo-Scientific, Philadelphia, PA, USA). The positive control group was exposed to 0.1% DMSO. Negative controls were treated with 100 ng/mL unlabeled recombinant human EGF (Cat#: E9644, I3390, MilliporeSigma, Saint Louis, MO, USA). Cells were then washed with pre-warmed PBS and incubated at 37 °C in serum-free IMD medium for 60 min, then fixed using a 1:1 methanol:acetone solution at −20 °C for 20 min, followed by three TBS washes. Cell nuclei were stained with DAPI (1:1000). For EGF endocytosis quantification, three random images (40X magnification) per well for a total of 3 wells per treatment group were obtained using (Lionheart FX, BioTek Instruments Inc, Winooski, VT, USA). Filter sets for 350 and 590 nm were used for detection of DAPI and Alexa Fluor Texas Red-conjugated EGF, respectively. Over 1800 cells from randomized fields from each group were analyzed using the software CellProfiler [67 (link)]. Total labeled EGF was normalized to the total number of nuclei.