Inverted microscope

For the wound healing scratch assays, a uniform wound was made by scratching with a 200 µL pipette tip when the transfected cells reached 90% confluence in 12-well plates. Cells were maintained in a serum-free culture medium after being washed three times with PBS. After 24 h, each well was photographed under an inverted microscope (Leica, Germany) at 100× magnification. The cells protruding from the border of the scratches were counted to calculate the wound recovery rate.
For the Transwell assays, Transwell inserts (Corning, USA) were pre-coated with 20 μg/μL of Matrigel (BD Biosciences, USA), then placed in a 24-well plate. Transfected cells were resuspended at a density of 3 × 10⁴/ml in serum-free culture medium and transferred to the upper chambers. In parallel, culture medium containing 10% FBS was added to the lower chamber of each well. After incubation for 24 h, cells on the inner membrane of the upper chamber were removed with cotton swabs. Invading cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.5% crystal violet for 20 min. Three fields of invading cells in each well were captured randomly and counted under an inverted microscope (Leica, Germany) at 100× magnification.

Calcification in the mouse aorta was observed using Von Kossa staining kit (ab150687, Abcam, UK). Briefly, aortic tissue sections were incubated with 5% silver nitrate solution for 30 min under a UV lamp and then treated with 5% sodium thiosulfate. Subsequently, the sections were counterstained using Nuclear Fast Red solution. Ca deposits were stained brown-black and observed under an inverted microscope (Leica, Germany).
Calcification of the MOVAS cells was observed using the Alizarin Red S staining method. Briefly, the cells were fixed in 4% formaldehyde for 5 min at 25°C, exposed to 2% Alizarin Red S (A5533, Sigma) for 30 min, and washed with 0.2% acetic acid. Calcified nodules were stained red and observed under an inverted microscope (Leica).
The Ca content in the aortic tissue section and cells were measured using the o-cresolphthalein coplexone method, as previously described [22 (link)]. Quantification of Ca (μg/mg protein) was normalized to the protein concentration, which was determined using the Bicinchoninic Acid Protein Assay Kit (P0012S, Beyotime).

Scratch healing assay: 8 × 10⁵ cells were inoculated into each well of a 6-well plate with a horizontal line at the bottom for 24 h at 37 °C in a 5% CO₂ incubator. After scratching with a 10 µL pipette tip perpendicular to the horizontal line at the bottom of the bottle, the old medium was replaced with serum-free medium, and culture was continued for the preset time. Observation and photographing were performed under an inverted microscope (Leica, Germany).
Transwell assay: When the cells had grown to cover 80% of the bottom area of the culture flask, they were starved in serum-free medium for 24 h. The cells were trypsinized to prepare a serum-free cell suspension at a density of 1 × 10⁶ cells/mL, and 100 µL of cell suspension were added to the upper chamber. Then, 600 µL of complete culture medium containing 10% serum were added to the lower chamber for 48 h at 37 °C in a 5% CO₂ incubator. The medium was discarded, and the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with 0.1% crystal violet for 10 min. Cell counting and photographing were performed under an inverted microscope (Leica, Germany).

RSC96 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and were incubated under normal glucose (5.5 mmol/L glucose) or high glucose (50 mmol/L glucose) conditions for 48 h. Subsequently, a pipette tip was used to scratch the cell layer, and the migration area was calculated 36 h later.
L929 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and were incubated under normal glucose (5.5 mmol/L glucose), high glucose (50 mmol/L glucose) or high glucose added with TGF-β1 (10 ng/ml) conditions for 48 h. Subsequently, the cell layer was scratched using a pipette tip, and the migration area was calculated 24 h later using a Leica inverted microscope.
L929 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and were incubated with or without RSC96 cell culture supernates for 48 h. Subsequently, the cell layer was scratched using a pipette tip, and the migration area was calculated 24 h later using a Leica inverted microscope.

Wound healing and transwell chamber assays were carried out to assess migration and invasion properties, as we previously described (9). For wound healing assay, 2 × 10⁵ transfected cells were plated in 12-well plates and allowed to grow till confluency. Next, a sterile pipette tip was employed to introduce a wound on the cell surface, before incubation in serum-free medium over 24 h before imaging at 0 and 24 h with an inverted microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA). For transwell invasion assay, 24-well Transwell chambers with polycarbonate filters (8-μm pores; Corning Inc.) were applied. Plasmid incorporated cells (1×10⁴ cells per well) in zero serum medium were introduced to the top chamber coated with Matrigel, and medium with 10% FBS was included in the bottom chamber for chemoattraction. After 24-h, the migrated cells were fixed with 4% formaldehyde for 20 min and then stained with 0.1% crystal violet for 5 min. Cell invasion was quantified with an inverted microscope (Leica Microsystems, Inc.) by choosing 5 random vision field per treatment.

For sphere formation of PT and PTU cells, cells were plated at a density of 20,000 cells/well in a 24-well ultra-low attachment plate (Corning, Corning, NY) in DMEM containing 2% Matrigel (growth factor–reduced, phenol red-free; Corning) and 10% FBS (Thermo Fisher) for 5–7 days at 37 °C in humidified air containing 5% CO₂. For the single-cell-derived sphere-forming assay under Matrigel dome-embedding conditions, 5,000 PT or PTU cells were suspended in 50 µl Matrigel and were carefully dispensed as droplets into a pre-warmed 24-well ultra-low attachment plate. After the Matrigel matrix dome was solidified in a humidified incubator at 37 °C for 15 min, 1 ml of warm DMEM with 10% FBS was added and cells were cultured for 7–10 days. The culture medium was changed every 2–3 days. Cells were observed daily, and spheres were counted under an inverted microscope (Leica Microsystems). To calculate the forming efficiency, spheres with diameters >50 µm were scored under an inverted microscope (Leica Microsystems). The sphere-forming efficiency (%) = scored sphere number/total plating cells. The area of each sphere was analyzed using ImageJ.