Calnexin

The collected NP tissues was ground to powder in liquid nitrogen. The cells at 24 h after treated with mechanical stress or above tissues powder were placed in RIPA lysis buffer (Beyotime, China) supplemented with 1 mM PMSF (Beyotime, China) on ice for 30 min. The collected liquid was centrifuged at 12,000 rpm for 15 min at 4 ℃. The protein concentration was detected with a BCA protein assay kit (PC0020, Solarbio). The protein samples from each group were separated in 8%, 10%, or 12% SDS–polyacrylamide gels (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with QuickBlockTM Blocking Buffer (Beyotime, China) for 20 min at room temperature, the membranes were incubated with rabbit primary antibodies against ACSL4 (1:2000, Proteintech), Bip (1:2000, Proteintech), Piezo1 (1:1000, Proteintech), Calnexin (1:5000, Proteintech), GPX4 (1:1000, Proteintech), ATF6 (1:2000, Proteintech), PERK (1:1000, Proteintech), Aggrecan (1:1000, Proteintech), Col-2 (1:1000, Novus), ADAMTS-5 (1:1000, Abcam), MMP-13 (1:1000, Proteintech), SelK (1:500, Proteintech), GAPDH (1:5000, Proteintech) overnight at 4 ℃. Then, the membranes were incubated for 90 min at room temperature with secondary antibody. The bands were visualized using an Amersham Imager 600, and the density was quantified using ImageJ software.

Cells and exosome samples were lysed in radioimmunoprecipitation assay buffer and boiled for 10 min. Lysate proteins were separated on a 12% polyacrylamide gel by electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA). The membranes were blocked in 3% non-fat dry milk for 1 h at room temperature (approximately 25 °C) and subsequently incubated with primary antibodies against CD63 (1:1000 dilution; Abcam, Cambridge, UK), TSG101 (1:1000 dilution; Abcam), calnexin (1:500 dilution; Proteintech Group, Rosemont, IL, USA), SOCS3 (1:500 dilution; Proteintech Group), and β-tubulin (1:2000 dilution; Proteintech Group) at 4 °C overnight. The membranes were washed three times with 0.2% Tween 20 in PBS following incubation with alkaline phosphatase-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL, USA).

The total protein contents were extracted from the HUVECs-derived exosomes, HUVECs, and cardiac tissues. Western blotting was performed following the standard procedures [24 (link)]. The following primary antibodies were used: Calnexin (1:2000, Proteintech, Rosemont, USA), tumor susceptibility gene 101 (TSG101, 1:500, Proteintech), CD63 (1:1000, Abcam, Cambridgeshire, UK), CD81 (1:1000, Abcam), B Cell Lymphoma-2-associated X protein (Bax, 1:5000, Proteintech), Bcl-2 (1:1000, Proteintech), cleaved caspase-3 (1:1000, Abcam), PI3K (1:5000, Proteintech), phosphorylated phosphatidylinositol 3-kinase (p-PI3K, 1:500, Abcam), AKT (1:5000, Proteintech), phosphorylated protein kinase B (p-AKT, 1:2000, Proteintech), and β-actin (1:5000, Proteintech). The following secondary antibodies were used: Goat anti-rabbit antibody (1:3000, Ant Gene, Wuhan, China) and Goat anti-mouse antibody (1:3000, Ant Gene).

Macrophages and cultured media were lysed in PBS or RIPA buffer containing protease inhibitor cocktail and were separated by native non-denaturing or denaturing 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blotted with antibody specific to AAT (DAKO, Carpinteria, CA), Ik $\beta$ , p-Ikk $\beta$ and $\beta$ -actin (Cell Signaling, Danvers, MA), Calnexin, CD63, TSG101, TNF- $\alpha$ , IL-1 $\beta$ (Proteintech, Chicago, IL), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA).

The collected NP tissues was ground to powder in liquid nitrogen. The cells at 24 h after treated with mechanical stress or above tissues powder were placed in RIPA lysis buffer (Beyotime, China) supplemented with 1 mM PMSF (Beyotime, China) on ice for 30 min. The collected liquid was centrifuged at 12,000 rpm for 15 min at 4 ℃. The protein concentration was detected with a BCA protein assay kit (PC0020, Solarbio). The protein samples from each group were separated in 8%, 10%, or 12% SDS–polyacrylamide gels (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with QuickBlockTM Blocking Buffer (Beyotime, China) for 20 min at room temperature, the membranes were incubated with rabbit primary antibodies against ACSL4 (1:2000, Proteintech), Bip (1:2000, Proteintech), Piezo1 (1:1000, Proteintech), Calnexin (1:5000, Proteintech), GPX4 (1:1000, Proteintech), ATF6 (1:2000, Proteintech), PERK (1:1000, Proteintech), Aggrecan (1:1000, Proteintech), Col-2 (1:1000, Novus), ADAMTS-5 (1:1000, Abcam), MMP-13 (1:1000, Proteintech), SelK (1:500, Proteintech), GAPDH (1:5000, Proteintech) overnight at 4 ℃. Then, the membranes were incubated for 90 min at room temperature with secondary antibody. The bands were visualized using an Amersham Imager 600, and the density was quantified using ImageJ software.

RAW264.7 cells were induced to an M1/M2 type for 24 h and cultured in an exosome-depleted FBS-containing complete medium. The supernatant was collected, and exosomes were obtained through ultracentrifugation [35 (link)]. Briefly, the supernatant was subjected to a series of differential centrifugation steps (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min) to remove intact cells and cell debris. Subsequently, the supernatant was centrifuged at 100,000×g at 4 °C for 70 min to isolate the proteins-containing exosomes. The exosomes were then purified by washing them with PBS and subjected to an additional centrifugation step at 100,000×g for 70 min.
The protein content of the exosomes was measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China) referring to the manufacturer’s instructions. CD9, CD63, CD81, TSG101 and Calnexin (Proteintech, Chicago, USA), which were exosomal marker proteins, were assessed by western blotting as previously described [51 (link)]. M2 macrophage-derived exosomes were scanned using a TEM (JEM1400, Tokyo, Japan). The size and concentration of exosomes were analyzed through NTA (Nanosight NS300, Malvern, UK).

Exosomes were analyzed by transmission electron microscopy using negative staining. A drop of exosomes (about 10 μL) was added on copper grid for 1 min, dried at 65 °C and observed on a HT7700 transmission electron microscope (HITACHI, Japan) equipped and operated at an acceleration voltage of 80 kV. Images were taken using a Gatan CCD (Gatan, Inc., US). Exosome purity was assessed by western blot analysis. Total cellular and exosomal proteins were respectively extracted from cells and exosomes using SDS lysis buffer (250 nM Tris-HCl, pH 7.4, 2.5% SDS). Proteins (10 mg/mL) were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane. The antibodies used for CD63, TSG101, calnexin and P-gp (Proteintech Group, CHI, USA), and enhanced chemiluminescence (ECL) plus kit (Millipore, America) was applied for visualization. The size distribution was detected by a nano-ZS90 analyzer (Malvern, Worcestershire, UK) after diluted 10 times.