Aa128

H22 or Bel7402 cells were treated with 200 μg mL⁻¹ PSiNPs for 6 h. After washing with PBS for three times, cells were lysed in RIPA lysis buffer and then subjected to western blot analysis. Briefly, 200 μg of lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 15% gel) and transferred onto nitrocellulose membranes. The membranes were blocked by 5% BSA for 2 h, and then incubated with anti-LC3 (Novus, NB100-2331SS) and anti-β-actin antibody (Beyotime, AA128, diluted to 1:2,000) at 4 °C overnight. After washing with Tris-buffered saline containing 0.1% Tween-20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Beyotime, A0216, A0208, diluted to 1:10,000) at 37 °C for 2 h. The protein bands were detected using enhanced chemiluminescence (ECL) reagent and analyzed on ChemiDoc XRS Gel image system (Bio-Rad, Hercules, CA, USA). Uncropped gel images are provided in Source Data file.
Bel7402 cells were transfected with EGFP-LC3 plasmid by electroporation. After 24 h transfection, the cells were treated with 200 μg mL⁻¹ PSiNPs for 6 h, washed with PBS for three times and then fixed with 4% paraformaldehyde. Green fluorescence of LC3 proteins were visualized by FV1000 confocal microscope (Olympus, Japan).

Proteins of A549, PC9 and TE1 cells after RNA interference were extracted using RIPA (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktail (Beyotime). The steps and the reagents used for measuring protein concentration and western blot are as described in previous studies [21 (link)].
Finally, we observed the protein bands with the Moon chemiluminescence kit (Beyotime). The following antibodies were used: Rabbit anti-CAV2 (CY5010, dilution 1:1500, Abways); Rabbit anti-PHLDA1 (AY3597, dilution 1:1500, Abways); rabbit anti-VDAC3 (55260-1-AP, dilution 1: 1500, Proteintech), mouse β-ACTIN (1:3000, AA128, Beyotime), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) (1:3000, A0208, Beyotime), and HRP-labeled goat anti-mouse IgG (H + L) (1:3000, A0208, Beyotime).
All the samples were repeated 3 times.

After being washed with cold PBS (Gibco, Grand Island, NE), cells were lysed in 200 μl RIPA lysis buffer (Beyotime Biotechnology, Shanghai, CN) containing 1% phenylmethanesulfonyl fluoride (PMSF; New Cell & Molecular Biotech, Suzhou, CN) and were vortexed briefly and centrifuged at 20,000 g for 10 min, 4°C. The supernatant was collected and protein concentration was determined using a Bradford protein assay kit (Bio-Rad, California, USA). Equal quantities of proteins (30 μg per well) were subjected to SDS-PAGE gels (10%) and transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA, USA) membranes. PVDF membranes were incubated in Tris-Buffered Saline-Tween (TBS-T, 10 mM Tris-HCl, 100 mM NaCl, and 0.5% Tween-20) with 5% non-fat milk for 1 h and then incubated with diluted antibodies to ChemR23 (1:1,000, ab64881, Abcam, Cambridge, UK) and β-actin (1:1,000, AA128, Beyotime Biotechnology, Shanghai, CN) at 4°C overnight. After washing, membranes were blocked with 5% non-fat milk in TBS-T and probed with peroxidase-conjugated secondary antibody to rabbit or mouse (BL003A and BL001A, Biosharp, Hefei, CN), respectively (1:2,000). The immunostainings were detected with an ECL kit (Millipore Sigma, Billerica, USA) and chemiluminescence captured by Gene Tools software (Syngene, Cambridgeshire, UK).

The total protein was collected using a protein extraction kit (Sigma-Aldrich, USA). The protein concentrations were determined using the BCA assay (Pierce, Thermo Fisher, USA). After denaturing at 95 °C for 5 mins, 5 mg aliquot of total protein of each sample was subjected to the 4–20% gradient SDS-PAGE, transferred to a PVDF membrane (Millipore, USA). The membrane was incubated in blocking solution with donkey serum (GTX30972, GeneTex, USA) for 1 hour, and was incubated overnight at 4 °C with the primary antibodies against Col X (ab58632, Abcam, USA), Col I (ab6308, Abcam, USA), Col II (ab116242, Abcam, USA), Sox 9 (PA5-23383, ThermoFisher, USA), Aggrecan (MA3-16888, ThermoFisher, USA), Notch1-ICD (07–1232, Millipore, USA), MMP-13 (MAB13426, Millipore, CA), Runx2 (sc-10758, Santa Cruz, USA) and β-actin (AA128, Beyotime, China). One of the following secondary antibodies was used: HRP-labeled goat anti-rabbit IgG (A0208, Beyotime, China) and HRP-labeled goat anti-mouse IgG (A0216, Beyotime, China). Proteins in the blots were visualized by an ECL plus kit (P0018, Beyotime, China). The integrated optical density of bands was quantified with the ImageJ software. Each sample was normalized to β-actin.

4T1 cells irradiated with a 4-Gy X-ray and incubated for 6 and 12 h were collected and processed with RIPA lysis and extraction buffer (89,900, Thermo Fisher Scientific). The cell extracts were denatured and resolved via 10% SDS-PAGE. A primary anti-Caveolin-1 rabbit monoclonal antibody (Abcam, ab32577, 1:200, Shanghai, CHN) and a goat anti-rabbit secondary antibody (Abcam, ab205718, 1:1000) were employed to detect Caveolin-1. β-actin determined by a mouse monoclonal antibody (AA128, Beyotime, 1:1000) was used as a reference. This assay was repeated three times on different days.

Aliquots of total kidney homogenate from individual animals were diluted in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 0.1 mM EGTA, 5 mM NaF, 1 mM Na₃VO₄, 5 mM Na₂PO₄ and 1 × proteinase inhibitor cocktail (Beyotime Institute of Biotechnology, China) to a final protein concentration of 2 μg/μL. Western blot analysis was conducted and quantified as described by Müller et al. [28 (link)]. The following antibodies were used: rabbit anti-FKN antibody (ab25088, Abcam Ltd, Hong Kong, 1:100 dilution) or anti-NFκB p65 antibody (ab31481, Abcam Ltd, Hong Kong, 1:200 dilution) or anti-NF-κB phospho p65 antibody (ab28810, Abcam Ltd, Hong Kong, 1:100 dilution) to evaluate the activity of NF-κB pathway, and mouse anti-beta actin monoclonal antibody (AA128, Beyotime Institute of Biotechnology, China, 1:500 dilution) followed by the goat polyclonal secondary antibody to rabbit IgG-H&L (HRP) (ab6721, Abcam Ltd, Hong Kong, 1:1000 dilution) or anti-mouse IgG-H + L (A0216, Beyotime Institute of Biotechnology, China, 1:1000 dilution). The image of western blots was scanned by Quantity One software and the original intensity of each specific band was quantified with freeware image analysis software, NIH Image (National Institute of Health, Bethesda, Md., USA).