Anti collagen 6

Cells from three donors at confluence (day 11) were used for each study. The cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) at RT for 30 minutes and permeabilized with 0.5% Triton X-100 in PBS for 30 minutes. After blocking with 2% goat serum for 2 hours at RT, the tissues were incubated overnight at 4°C with primary antibodies anti-ZO-1, 1 : 200 (ZO1-1A12, Thermo Fisher Scientific, Rochester, NY, USA); anti-Ki-67, 1 : 200 (MIB-1, Milan, Italy); and anti-vinculin, anti-collagen VI, and anti-laminin I, 1 : 200 (Abcam, Cambridge, Massachusetts, USA). The samples were incubated with goat anti-mouse fluorescein isothiocyanate- (FITC-) conjugated secondary antibody in 20% goat serum for 2 hours at RT. After each step, the cells were washed 3 times with 1x PBS and covered with mounting medium and cover slips. Samples were examined with the LSM 510-metalaser scanning microscope (Zeiss, Milan, Italy).

Cells were lysed in lysis buffer, and the total protein concentration was determined with the BCA Protein Assay Kit (Beyotime, cat# P0011). Equal amounts of protein samples were electrophoresed by SDS–PAGE and then transferred to PVDF nitrocellulose membranes. The membranes were blocked in 5% BSA at room temperature for 1 h and incubated with the following specific primary antibodies: anti-SIX1 (1:1000, CST, cat# 16960), anti-collagen VI (1:1000, Abcam, cat# ab182744), anti-TGF beta receptor II (1:1000, Abcam, cat# ab269279), anti-Smad2/3 (1:1000, CST, cat# 5678), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (1:1000, CST, cat# 8828), and anti-β-ACTIN (1:1000, CST, cat# 4970). The next day, the membranes were washed with TBST three times and incubated with an anti-rabbit IgG HRP-linked antibody (1:2000, CST, cat# 7074) at room temperature for 50 min. The membranes were imaged using a ChemiDoc XRS + system (Bio–Rad, USA). To further confirm the involvement of TGF-β signaling in mechanical SIX1-induced collagen expression, cells were pretreated with or without mTGF-β1 (50 ng/ml, CST, cat# 5231LF) for 2 or 4 h.