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Furthermore, LTBP-1 and fibronectin co-localisation was significantly reduced with ATRA treatment compared to vehicle treated PSCs (p Preparation of LTBP-1 rich ECM and LTBP-1/fibronectin co-localisation by PSCs (a) HEK-293 were seeded on coverslips and transfected with LTBP-EGFP.
After 7 days of culture, HEK-293 cells were removed with DOC buffer and replaced by PSCs which secrete fibronectin (FN) into the HEK-293-generated ECM.
We first generated an LTBP-1-rich ECM by allowing LTBP-1-transfected HEK-293 cells to deposit ECM on glass coverslips for 7 days (Fig. The HEK-293 cells were then removed with sodium deoxycholate (DOC) buffer as described in the materials and methods and in Fig. We used HEK-293 cells because they deposit minimal fibronectin and they were not able to organise LTBP-1 into fibrils (Fig. After removing HEK-293 cells, untreated PSCs (hereafter control) or 10 day ATRA (1 μM) pre-treated PSCs (hereafter ATRA) were seeded on matrices and cultured for a further 2 days, during which time PSCs secrete and incorporate fibronectin into the ECM.
Endogenous expression of LTBP-1 by PSCs was not detectable during 48 hours either on glass coverslips or HEK-293 deposited ECM (Fig. Coverslips were then dual-stained for LTBP-1 and fibronectin.