Vimentin

The cell morphology and surface protein expression of the two types of cells were compared. Morphologic observations of the third passage dADSCs and VFFs were made using an inverted microscope. The cell surface protein expression levels were identified by cell immunofluorescence.
When the cells reached 80% to 90% confluence, they were fixed in ice-cold 4% paraformaldehyde for 10 minutes and rinsed with phosphate-buffered saline (PBS). They were permeabilized in PBS containing 0.3% Triton X-100 for 30 minutes at room temperature (RT). All cells were blocked (3% horse serum in PBS, 30 minutes, RT) and incubated for 2 hours (4°C) with the following primary antibodies: vimentin (monoclonal mouse vimentin, 1∶200; Novus, Littleton, CO, USA) and fibronectin (polyclonal rabbit fibronectin, 1∶100; Abcam, Cambridge, MA, USA). After a thorough washing, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1∶400; Molecular Probes, Eugene, OR, USA) and 594-conjugated goat anti-mouse IgG (1∶400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour (RT). All slides were counterstained using Hoechst 33342 (1∶1000; Roche; 10 minutes, RT) for nuclear staining.

Cells were grown on 6-well plates at a seeding density of 2.5 × 10⁵ cells per well. These cells were starved in serum-free RPMI-1640 for 24 h. They were then treated with 5 ng/mL TGFβ and 20 µM GSK-J4, both individually and in combination. After 48 h of treatment, the cells were scraped in RIPA lysis buffer for protein extraction. Protein concentration was estimated using the Bradford assay. SDS-PAGE and Western blot were carried out as per standard protocol. Primary antibodies used were p-SMAD3 (Santa Cruz, Dallas, TX, USA), N-cadherin (Novus, St. Louis, MO, USA), vimentin (Novus, St. Louis, MO, USA), H3 (Invitrogen, Waltham, MA, USA), H3K9me3 (Santa Cruz, Dallas, TX, USA), H3K4me3 (Invitrogen, Waltham, MA, USA), H2K27me3 (Abcam, Cambridge, UK), and actin (Invitrogen, Waltham, MA, USA), and the secondary antibodies were Chicken Anti-Mouse IgG H&L (HRP) (Abcam, Cambridge, UK) and Chicken Anti-Rabbit IgG H&L (HRP) (Abcam, Cambridge, UK). The blot was developed using an ECL reagent (Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate, Waltham, MA, USA). The experiment was repeated thrice. The statistical analysis was carried out using ImageJ 1.52a software. The student’s t-test was carried out to calculate significance using GraphPad Prism 9.5.0 software.