Imagej software version 6.0

Renal tissues were fixed in 4% paraformaldehyde, dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol, and embedded in paraffin. Paraffin-embedded tissues were sectioned at 4 μm, rehydrated in a series of xylene and ethanol solutions, and then used for Masson Trichrome staining, which stains normal tissue regions red, nuclei black, and fibrosis regions blue. Observations of histopathological changes in kidney tissues were performed by light microscopy (Leica, Nusslock, Germany). Eight fields per sample were randomly selected for fibrotic area quantification using Image J software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Images were acquired under identical conditions at the same magnification. The fibrotic area was expressed as the percentage of the captured image area.

C. muridarum-infected mice lung tissues were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors, and protein concentrations were determined using a BCA (Thermo Fisher Scientific, Rockford, IL, USA) kit. Subsequently, the membrane was placed in a 5% blocking buffer with bovine serum albumin for 1 h, incubated with a primary antibody against caspase-3 (Proteintech, Chicago, IL, USA), GSDMD (Cell Signaling Technology, Danvers, MA, USA), GSDME (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Abcolonal, Shanghai, China) at 4 °C overnight, and then incubated with horseradish peroxidase-labeled secondary antibodies (Absin, Shanghai, China) at room temperature for 1 h. Finally, an enhanced chemiluminescence kit was used for visualization. The collected images were examined with ImageJ software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

HRECs were lysed with RIPA lysis buffer (Sangon Biotech, Shanghai, China) containing protease inhibitors and phosphatase inhibitors, and the protein concentrations were determined using the BCA kit (Thermo Fisher Scientific, Inc.). The samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 140 V and transferred to polyvinylidene membranes. Subsequently, the membranes were placed in 5% blocking buffer (bovine serum albumin) to block for 1 h, incubated with primary antibodies against CASP3 (Abcam, MA, USA), GSDMD (Abcam, MA, USA), GSDME (Abcam, MA, USA), and GAPDH (Abcam, MA, USA) at 4˚C overnight, followed by horseradish peroxidase-labeled secondary antibodies (Abcam, MA, USA) for 1 h at room temperature, and finally developed with an enhanced chemiluminescence kit for visualization. The collected images were examined using ImageJ software (version 6.0; Media Cybernetics, Inc.).