Pre stained protein standard

The protein concentrations of isolated EVs were determined using Bradford protein assays (Sangon Biotech, Shanghai, China). Proteins (1.5 μg) from isolated S. japonicum EVs were separated using precast 4–20% polyacrylamide linear gradient gels (Bio-Rad, Hercules, CA, USA). A pre-stained protein standard (Thermo Scientific, Waltham, MA, USA) was used to track protein migration. The gels were sliver-stained as previously described [8 (link)] and scanned using a Bio-Rad Molecular Imager FX system (Bio-Rad).

For Western blot analyses, proteins were isolated 72 h after transfection as previously described [62 (link)]. For electrophoresis, a 4% to 20% precast gradient gel (Bio-Rad Laboratories, Feldkirchen, Germany) was loaded with 30 µg of whole protein extract and 15 µg of a prestained protein standard (ThermoFisher Scientific, Waltham, MA, USA) for molecular weight evaluation. Electrophoresis, protein transfer, and antibody staining were performed as previously described [10 (link)]. Incubation with the primary antibody was performed overnight at 4 °C and with the secondary antibody for two hours at room temperature. Antibody details are specified in Supplementary Table S4. Chemiluminescence of bound antibodies was induced using ECL substrate (ThermoFisher Scientific, Waltham, MA, USA) and visualized on X-ray films. Afterwards, the membrane was stripped and then stained again to visualize another protein of interest. The intensity of specific bands was measured with ImageJ Ver. 1.53 (NIH, Bethesda, MD, USA) and normalized to tubulin as the reference protein. Importantly, samples and respective controls were stained on the same gel.

The phage structural proteins were analyzed using the method previously described by Lu et al. (2003) (link) with some modifications. Briefly, the ultracentrifuge-purified phage particles were mixed with SDS-PAGE sample buffer and then heated in a boiling water bath for 10 min. The boiled sample was loaded onto a NuPAGE precast gradient minigel (4–12% Bis-Tris, Invitrogen Corporation, Carlsbad, CA, USA). Electrophoresis was carried out at 75 V for 2 h. Pre-stained protein standard (Invitrogen) was used to estimate the molecular weights of the proteins. The gel was stained with SimplyBlue SafeStain (Invitrogen).

Culture supernatant and recombinant proteins (AAT (Athens Research), SLPI (R&D Systems), elafin (Proteo Biotech)) were quantified by bicinchoninic acid assay and electrophoresed on 12.5% SDS-polyacrylamide gels. For band size determination the protein marker SeeBlue® (Thermo Fisher Scientific: Waltham, MA, USA) Pre-stained Protein Standard (Invitrogen, LC5625) (Thermo Fisher Scientific: Waltham, MA, USA) or Mark12^TM Unstained Standard (Invitrogen, Bio Sciences Ltd, Ireland) was used. For Coomassie blue silver staining, gels were fixed in ethanol (50% v/v) and phosphoric acid (2%) for one hour and washed × 3 in dH2O prior to staining with Coomassie blue silver overnight. For western blotting, following SDS-PAGE, proteins were transferred to PVDF and signals were detected using goat anti-AAT (Abcam) (https://www.biocompare.com/), goat anti-SLPI (R&D Systems) or mouse anti-elafin (Abcam) with appropriate secondary antibodies and Immobilon Western chemiluminescent HRP substrate (Millipore) using the Syngene G:Box Chemi XL gel documentation system (Syngene International Limited, New Delhi, India).

Recombinant human TNF-α (Sino Biological Inc., China); curcumin (Sigma-Aldrich, St. Louis, MO, USA); pre-stained protein standards (Fermentas, Lithuania); Human Annexin V Apoptosis Detection Kit and cell cycle test kits (BD Biosciences, San Jose, CA); Bradford Assay kit (BD Biosciences, San Jose, CA). Antibodies against NF-κB-p65, phospho-NF-κB-p65, caspase-8, caspase-9, ERK, phospho-ERK (pERK), Akt andphospho-Akt (pAkt) (Cell Signaling Technology, Boston, MA, USA); β-actin antibody (Santa Cruz Biotechnology, USA); Nuclear and Cytoplasmic Protein Extraction Kit, LDH Cytotoxicity Assay Kit and CCK-8 Assay Kit (Beyotime, CHN); ECL detection kit (Cell Signaling Technology, Boston, MA, USA). Water was ultra-pured by a Milli-Q water purification system (Millipore, USA).

Antibodies against cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, AKT, phospho-Akt (p-Akt), ERK, phospho-ERK (p-ERK), JNK, phosphor-JNK (p-JNK) (Cell Signaling Technology, Boston, MA, USA); anti-p62/SQSTM1(MBL); antibody against LC3B, Curcumin, 3-methyladeine (3-MA), chloroquine diphosphate (CQ), Rapacymin, the fluorescent dye Acridine Orange (AO) and Crystal violet (Sigma-Aldrich, St. Louis, MO, USA); Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA); pre-stained protein standards (Fermentas, Lithuania); Human Annexin V Apoptosis Detection Kit and cell cycle test kit (BD Biosciences, San Jose, CA); β-actin antibody (Santa Cruz Biotechnology, USA); Senescence β-Galactosidase Staining Kit (Beyotime Biotechnology). Cur was dissolved with dimethyl sulfoxide (DMSO) and the storage concentration was 20 mM/ml; Rapacymin was solved in DMSO; 3-MA, CQ and AO were solved in ultrapure water.