Laminin 521

Undifferentiated podocytes were seeded onto a 96-well IncuCyte® ImageLock microplate coated with 0.25 μg/cm² laminin 521 (CORNING) at a density of 40,000 cells per well. After cells reached a confluent state, they were serum starved in RPMI containing 1% FBS for 2 h at 37 °C. A scratch wound was made using the IncuCyte 96-well Wound Maker (Sartorius). Cells were then kept under non-permissive conditions up to 24 h and the migration rate was analyzed by IncuCyte Analysis Software (Sartorius).

Undifferentiated podocytes were plated on glass cover slips coated with 0.25 μg/cm² laminin 521 (CORNING) and allowed to differentiate for 7 days. They were then fixed in 4% PFA before being permeabilized with 0.1% Triton. After blocking with 3% bovine serum albumin in PBS, cells were immunostained with the respective antibodies and stained with phalloidin. Immunofluorescence images were obtained using a Zeiss LSM780 laser scanning confocal microscope with the Zeiss Plan Apochromat 63 × 1.40 Oil DIC objective. All imaging parameters were maintained constant throughout image acquisition of all samples. Quantification of FA complex was done using ImageJ software, as described previously^{49 (link)}. Briefly, cell contour was traced using phalloidin-stained images with the ‘Freehand Line’ function and cell area was measured. After shifting to images with vinculin staining, the ‘Clear Outside’ function was used to erase the area outside the cell region. The number of particles between 1 and 8 μm² was counted. FAs were classified into three groups: small (1–2 μm²), medium (2–6 μm²), and large (6–8 μm²). The proportion of the number of FAs in each group relative to total FAs was calculated.