Rat tail collagen

The cell migration was measured using 8 μm pore polycarbonate membrane transwell chambers (BD Biosciences, Stockholm, Sweden) coated with 25 μg/ml rat tail collagen (Roche) as described before with some modifications [24 (link)]. Cells were starved in 0.5% DCC-treated FCS in phenol-free DMEM for 48h and then seeded at 25 000 cells/well each transwell chamber and let migrate towards vehicle (DMSO), 2 nM HRG, 10 nM E2 for 24h and/or 20 nM 4-OHT. The non-migrated cells were scraped from the top membrane using a cotton swab and the migrated cells were fixed in 4% para-formaldehyde and stained in 0.1% crystal violet (Sigma Aldrich). The cells were imaged using an Axiovert S100 inverted microscope (Carl Zeiss, Oberkochen, Germany). The migrated cells were counted in five different fields in duplicate wells, in at least three independent experiments and expressed as the mean ± S.E.M.
For proliferation assays, 25 000 cells/ml were let to adhere to the culture plate for 4 h in plain medium and then starved in phenol-free DMEM supplemented with 1% DCC-treated FCS for five days in the presence of vehicle (DMSO), 2 nM HRG, 10 nM E2 for 24h and/or 20 nM 4-OHT. Viable cells were counted using Trypan blue exclusion in at least three independent experiments and expressed as the mean ± S.E.M.

hSAECs were incubated in the absence or presence of TGFβ (10 ng/mL) for 15 days, re-plated on glass cover slips pretreated with rat tail collagen (Roche Applied Sciences) and fixed with 4 % paraformaldehyde in PBS. Afterwards, the fixed cells were stained with Alexa Fluor® 568 phalloidin for cytoplasmic distribution of F-actin (shown in red color) and also counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) for nuclear staining (shown in blue color). The cells were visualized with a Nikon fluorescence confocal microscope at a magnification of 63× [5 , 23 (link)].

Proteins localized in lamellipodia were purified, as previously described.40, 59 Briefly, cells were plated on 3 μm porous polycarbonate Transwell membrane inserts (Costar) that were coated on the bottom side with 25 μg/mL rat tail collagen (Roche, Inc.). The lower chamber contained medium with 0.5 nM HGF. Cells were allowed to extend their lamellipodia through the pores. Cell bodies remaining on the upper surface were removed and the lamellipodia extending to the lower surface were recovered in lysis buffer.