Gelatin

For immunostaining, cells were first fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and subsequently washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich). Next, cells were incubated in 0.1 M glycine (Sigma-Aldrich) for 5 min and then washed again with PBS. Triton-X was used for 2 min and then washed with PBS. For blocking, cells were incubated with 3 ml of 0.2% Gelatin (Sigma-Aldrich) solution for 3 h at room temperature. Primary antibody treatment was done with Recombinant Anti-Rab4 antibody (Abcam) at 1:200 dilution in Gelatin and kept overnight at 4 °C to mark recycling endosomes. Goat Anti -Rabbit IgG H&L, Alexa Fluor 488 secondary antibody (Abcam) was used at 1:500 dilution at Gelatin for 2 h after washing with PBS. Subsequently, cells were imaged in 2 ml of PBS.

AMCs were treated with 300 U/mL TNFα. After treatment, the cells were centrifuged, and condition media were collected. The protein concentration was evaluated by the Bradford assay (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy). A total of 40 µg of the non-reduced protein sample was loaded on 7.5% SDS-polyacrylamide gels containing 1 mg/mL gelatin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), following the gelatin Zymography Protocol developed by Abcam (gelatin-zymography-protocol">https://www.abcam.com/protocols/gelatin-zymography-protocol, accessed on 24 July 2023). Gels were stained with 0.5% Coomassie Blue R-250 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and destained in 10% acetic acid. Enzymatic activities appear as clear bands on a dark background. Equal amounts of cell lysate were loaded for the gelatin zymography test. The data obtained by densitometry for each band were referred to as the standard protein and expressed as relative peaks of the area by ImageJ software V1.52. The results were presented as histograms with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).