Widefield microscope

The anti-senescence activity of the BNC-G-HDE2 membrane was evaluated using the β-galactosidase (β-gal) staining assay. The BNC-G membrane was used as control. To prepare the membranes extracts, sterilized samples (10 cm²) were incubated in 10 mL of complete DMEM medium as previously described for the cytotoxicity assay.
NIH/3T3 cells were seeded in 12-well plates at a density of 2.5 × 10⁴ cells/well and allowed to stabilize for 24 h. Subsequently, 12.5 µM etoposide was used to induce cellular senescence in NIH/3T3 for 24 h. Etoposide-stimulated cells were then treated with the BNC-G-HDE2 membrane extract for another 24 h. As control, non-treated cells were incubated with DMEM medium or with extracts of BNC-G and BNC-G-HDE membranes. After incubation, culture medium was removed, and cells were washed with PBS and marked with β-gal solution prepared as described by the manufacturer (Cell Signaling Technology, Danvers, MA, USA). The analysis of the positive cells for senescence was performed at 20× magnification using a widefield microscope (Carl Zeiss, Oberkochen, Germany). At least 3 independent experiments were performed in replicate, and the percentage of β-gal-positive cells was determined using four microscopic images.

Collective migration assays were performed using 2-well silicon inserts (Ibidi). Glass coverslips were coated with fibronectin solution at 20 µg/mL in water, for 1 hr at room temperature. The cover slip surface was washed with sterile water and air-dried. Ibidi inserts were deposited on the fibronectin coated surface and 40,000–50,000 cells were loaded per well. For fluorescence live imaging, cells were directly plated after transfection in the well. After 3–4 hr of incubation, cell division was blocked using mitomycin at 10 µg/mL in CaCo-2 culture medium, for 1 hr. After overnight incubation in a fresh CaCo-2 culture medium, the insert was removed. Experiments were performed 24 hr to 48 hr after insert removal.
For individual cell migration, cell division was blocked using mitomycin at 10 µg/mL in CaCo-2 culture medium, for 1 hr. After overnight incubation in a fresh CaCo-2 culture medium, cells were detached and plated on a glass coverslip coated with 20 µg/mL fibronectin. After 6 hr, cells were imaged for 24 hr.
Collective and individual cell migration assays were performed using a Zeiss Wide-Field Microscope and imaged at 1 frame every 10 min. Cell trajectories were analysed using Manual Tracking from Fiji. Cell directionality was calculated as the ratio between the net displacement and the trajectory length within the last 20 hr of collective migration.

The human podocyte cell line was cultured in RPMI medium supplemented with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) supplement and 200 units/ml penicillin and streptomycin as described previously at 33 °C and 5% CO2^{31 (link)}. Podocytes were treated with puromycin aminonucleoside (PAN) (100 μg/ml) in the absence or presence of 1 µM ISD for a period of 48 hours. The cells were washed with PBS and fixed with 4% paraformaldehyde (in 1 × PBS), followed by permeabilization with 0.1% SDS. Immunostaining was performed using Neph1 (Alexa-488) and ZO1 Alexa-594) specific antibodies. Fluorescence microscopy was performed using a Zeiss wide-field microscope keeping all parameters constant, including exposure time while collecting images. The images were processed using Image J software and transferred to Adobe Illustrator for labeling. Brightness and contrast adjustments were kept constant throughout the images.

HaCaT and NIH/3T3 cells were seeded in 12-well plates at a density of 1.5 × 10⁵ or 2.5 × 10⁴ cells/well, respectively, and were allowed to adhere for 24 h. Thereafter, cellular senescence was induced with 100 µM etoposide (Sigma-Aldrich, St. Louis, MO, USA) for 72 h or 12.5 µM etoposide for 24 h in the HaCaT and NIH/3T3 cells, respectively. After the incubation period, the senescent cells and controls (exposed to etoposide-free medium) were treated in the absence or presence of 0.16 mg/mL EO or 0.8 μg/mL HRW for 24 h. Then, the culture medium was discarded, the cells were washed with PBS, fixed, and stained with freshly prepared β-galactosidase (β-gal) staining solution following the protocol provided by the manufacturer (Cell Signaling Technology, Danvers, MA, USA). Finally, the senescent cells were quantified using a widefield microscope (Carl Zeiss, Oberkochen, Germany) at a magnification of 40× by determining the percentage of β-gal-positive cells in randomly selected fields of four microscopic images, in at least three independent experiments performed in duplicate.