Qcmtm gelatin invadopodia assay

For quantitative analysis of pericellular proteolytic activity, the QCM^TM Gelatin Invadopodia Assay (Millipore) was applied according to the manufacturer’s instructions. Briefly, 10⁴ hMSCs were plated onto a thin uniform layer of fluorescein-labeled gelatin (green) affixed to the bottom of glass chamber 8-well slides (Ibidi, Martinsried, Germany). After incubation for 24 h in a cell incubator at 37 °C and 5% CO₂, degraded areas of gelatin, now devoid of fluorescence, were visualized by fluorescence microscopy. TRITC-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) were used for counterstaining of cytoskeletal F-actin and nuclei, respectively. Areas covered by cells and areas of degraded gelatin were quantified using ImageJ analysis software.

Invadopodial formation was evaluated using QCM^TM Gelatin Invadopodia Assay (Millipore, Billerica, MA, USA; cat No. ECM670) according to the manufacturer's instructions. Briefly, UM-UC-3 was transfected by NC siRNA, COL4A1 siRNA, or COL13A1 siRNA. After a 48-h incubation, cells were trypsinized, and 20,000 cells were seeded per well of a Lab-Tek 8-well chamber slide, which was pre-coated with FITC-labeled gelatin. After a 24-h incubation at 37°C, cells were fixed with 4% paraformaldehyde and stained with TRITC-phalloidin and DAPI for immunofluorescence microscopy (Leica DMI 4000B, Wetzlar, Germany). Invadopodia were visible as dark spots devoid of fluorescence. The number of formed invadopodia was counted and quantified for 20 cells in each group.

5 × 10⁴ cells were seeded on a 16 mm coverslip coated with a 6:1 mix of unlabeled:Cy3-labelled gelatin according to the manufacturer's instructions (QCM^TM Gelatin Invadopodia Assay; Millipore). After 18 h at 37°C, cells were fixed and stained for actin (Acti-stain^TM 670 phalloidin, Cytoskeleton). ECM degrading cells were defined as cells with an underlying black area depleted of fluorescent gelatin. Active cells were scored and expressed as percentage of the total cell population. In addition, the degraded area per cell was measured using the threshold tool of ImageJ software, and a degradation index corresponding to the ratio of degraded gelatin surface per cell was calculated.