Celld 3

To monitor the effect of the treatments on the cell phenotype, TK173 and TK188 cells were cultivated overnight in 8-well chamber slides coated with high-quality sterile polylysine (30,000 cell/well). The FCS-free cell culture and the cell treatment were performed as described above. After the treatments, the medium was removed, and the cells were washed twice with PBS buffer. Fixation of the cells was carried out for 20 min at 25 °C with 4% paraformaldehyde in PBS. Non-specific binding sites were blocked with 10% goat serum in 1% BSA (BSA/PBS) (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37 °C. The incubation with the primary antibodies was carried out overnight at 4 °C. Alexa Fluor 488-conjugated goat anti-mouse-antibody or Alexa Fluor 555-conjugated goat anti-rabbit-antibody were used as secondary antibodies. The incubation with the secondary antibody was performed for 60 min at 37 °C in the dark. The unbounded secondary antibody was removed with three successive wash steps with PBS-buffer for 10 min each. Thereafter, the samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in PBS buffer for 5 min. The monitoring of the staining was performed on an immunofluorescence Laser Scanning Cytometer IX71 (Olympus, Hamburg, Germany) using the CellD 3.4 software (Olympus, Hamburg, Germany).

To monitor the effect of the TGF-β1 or PDIA3, and the combined treatments on cell cycle, TK188 cells were cultivated overnight in 8-well Chamber slides (30,000 cell/well, Thermo Fisher Scientific). The cell treatments were carried out in FCS-free medium. After the treatments, the medium was removed, and the cells were washed twice with PBS buffer. Fixation of the cells was carried out for 20 minutes at 25°C with 4% paraformaldehyde in PBS. Nonspecific binding sites were blocked with 10% goat serum in 1% BSA (BSA/PBS) (Sigma-Aldrich) for 60 minutes at 37°C. The incubation with the primary anti-PCNA (catalog ab29 Abcam) and anti-cyclin D1 (catalog b134175 Abcam) antibodies was carried out overnight at 4°C. Alexa Fluor 488–conjugated goat anti-mouse antibody rabbit (catalog AB_2536161, Thermo Fisher Scientific) or Alexa Fluor 555–conjugated goat anti-mouse antibody (catalog AB_2536164, Thermo Fisher Scientific) were used as secondary antibodies. The incubation with the secondary antibody was performed for 60 minutes at 37°C in the dark. The unbounded secondary antibody was removed with 3 successive wash steps with PBS buffer for 10 minutes each. Thereafter, the samples were counterstained with DAPI in PBS buffer for 5 minutes. The monitoring of the staining was performed on an immunofluorescence Laser Scanning Cytometer IX71 (Olympus) using the CellD 3.4 software (Olympus).