Zli 9056

To evaluate the expression of HGF cell-adhesion-related proteins on the surfaces of different materials, specific immunostaining with VCL and ITGB1 was performed. HGFs were seeded on samples in 24-well plates, then fixed with 4% paraformaldehyde after 4 and 24 h of incubation, permeabilized with 0.1% Triton X-100, blocked with goat serum blocking solution (ZLI-9056, ZSGB-BIO, Beijing, China) for 1 h, and incubated overnight with specific primary antibodies targeting VCL or ITGBI (Abcam, Cambridge, UK). After washing, the membranes were incubated with the secondary antibody for 1 h in the dark. Actin staining was performed after incubation with Alexa Fluor 555 phalloidin (Cell Signaling Technology, Boston, MA, USA) for 1 h. Nuclear staining was performed using DAPI. Images were obtained using a confocal laser-scanning microscope (Leica, Hamburg, Germany).

RKO and HT115 cells were seeded onto coverslips placed into a 6-well plate at a concentration of 2 × 10⁴/mL, followed by incubation at 37 °C under 5% CO₂ humidified atmosphere for 48 h and then washing the adherent cells with sterile PBS thrice. Next, the PBS was discarded and 4% paraformaldehyde was added to each well for 20 min at the room temperature. The cells were washed three more times with sterile PBS, and the slides were blocked with methanol containing 3% hydrogen peroxide for 10 min. The samples were then rinsed thrice with PBS, permeabilized with 0.25% Triton X-100 (9002931; AMRESCO, USA) in PBS for 10 min, and blocked with 5% normal goat serum (ZLI-9056; ZSGB-bio) in PBS for 60 min. After blocking, the cells were incubated with the corresponding primary antibodies RUNX2 (ab76956; Abcam, UK) and BRG1 (21634–1-AP; Proteintech, USA) overnight at 4 °C, followed by treatment with anti-mouse-Alexa Fluor 594 and anti-rabbit-Alexa Fluor 488 secondary antibody, respectively, for 1 h at the room temperature. Next, 4′,6-diamidinophenyl-indole (DAPI) was used to stain the cell nuclei for 5 min, and the slides were observed under a confocal microscope (TCS-SP5; Leica, Mannheim, Germany). The specific antibody information is provided in Additional file 1. Table S1.

Brains were harvested following cardiac perfusion with ice-cold PBS and 4% PFA and dehydrated using 30% sucrose. All brains were cut into 30 µm thick coronal sections with frozen sections, and cells grown on cover glass were fixed with 4% PFA. All staining samples were permeated with 0.25% Triton X-100 (T109027, Aladdin) at room temperature. After washing the permeabilized cells three times with PBS, the sample was blocked with 0.25% Triton X-100 containing 10% goat serum (ZLI-9056, ZSGB-BIO). After that, the samples were incubated with primary antibodies overnight at 4 °C. Subsequently, the samples were washed three times and incubated with IgG (H + L) Alexa Fluor 488 goat anti-mouse (Invitrogen, A11001) or IgG (H + L) Alexa Fluor 594 goat anti-rabbit (Invitrogen, A11012) secondary antibody for 1 h at room temperature. Finally, the slices and cells were incubated with DAPI (Southern Biotech, USA, CAT No. 0100-20). Images were obtained (594/618 nm for the rabbit Dylight-594-conjugated antibody, 488/519 nm for the mouse Alexa Fluor 488-conjugated antibody) using fluorescence microscopy (A1 HD23, Nikon, Tokyo, Japan). A minimum of three coverslips were used for each experimental group. The area of ER of neurons was analyzed using Image J software according to a method in the previous study [29 (link)].

The aortic tissue sections were deparaffinized, rehydrated, and washed in distilled water. For immunohistochemical analysis, the sections were washed three times with PBS, fixed in 4% PFA for 15 min, and permeabilized with 0.1% Triton X-100 (T9284, Sigma, MO, USA) for 15 min. The sections were incubated with 3% H₂O₂ for 10 min and incubated with goat serum (ZLI-9056, ZsBio, Beijing, China) to block endogenous peroxidase activity. Then, the sections were incubated with GPX4 (1:200, ab125066, Abcam, USA) or p-AKT (1:200, 4060S, CST, USA) antibodies overnight at 4 °C. The following day, the sections were washed three times with PBS and incubated with goat anti-rabbit immunoglobulin gamma (IgG) secondary antibodies (ZF-0136, ZsBio, Beijing, China) for 30 min at room temperature. Positively stained cells were visualized by adding 3,3′-diaminobenzidine (GK600505, GeneTech, Shanghai, China).

Paraffin-embedded kidney sections were incubated at 60 °C for 60 min, deparaffined with xylene three times for 15 min each, hydrated with gradient ethanol solution for 5 min each, and finally immersed in deionized water. Following a 20 min incubation at 95 °C with an antigen retrieval solution for antigen retrieval, the sections were let to cool naturally to room temperature. The endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ at room temperature for 10 min. The sections were then blocked with goat serum (ZLI-9056, ZSBIO company, Beijing, China) at 37 °C for 30 min, followed by incubation with anti-nephrin (1:2000, ab216341, Abcam), anti-podocin (1:1000, ab50339, Abcam) and anti-cleaved caspase-3 (1:400, 19677-1-AP, Proteintech, Wuhan, China) antibodies overnight at 4 °C. The sections were then washed in phosphate-buffered saline (PBS), treated for 20 min at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibody (PV-9001, ZSBIO business), and colour development was performed by incubating with diaminobenzidine (DAB, ZLI-9018, ZSBIO company). Finally, the nuclei were stained with haematoxylin. At least five randomly chosen fields for each sample were evaluated under the microscope and analysed with Image-Pro Plus 6.0 software.

Details about the methods and further the semiquantitative assessment followed previous reports [7 (link)]. Briefly, 4 μm sections with different types of tumour tissues were dewaxed and rehydrated according to standard methods. Antigen retrieval was conducted with retrieval solution (ethylenediaminetetraacetic acid [EDTA], pH 9.0, ZLI-9069 from ZSGB-BIO, Beijing, China) for 4 min using a pressure cooker. H₂O₂ (PV-9000; ZSGB-BIO) was applied to block endogenous enzyme activity, and the samples were subsequently incubated in a serum-free blocking solution (ZLI-9056; ZSGB-BIO). Then, the primary antibody against OPN3 (MD4034-100; Medical Discovery Leader (MDL), Beijing, China) was diluted 1:300 at 4 °C overnight, followed by treatment with the UltraView Polymer DAB Detection Kit (Ventana/Roche) according to the recommended manufacturing protocol.
OPN3 expression on all stained slides was scored by two independent investigators. The semiquantitative assessment method was conducted by using percentages of 3 + (strong), 2 + (moderate), 1 + (weak), and 0 (negative) staining of tumour cells for each sample. The overall score was calculated by the percentage of positive tumour cells (3 × x % + 2× x % + 1 × x % = total score) to equal a range of 0-300 [21 (link)].

Briefly, paraffin-embedded sections (4 μm) were dewaxed in 100% (v/v) xylene and rehydrated in descending concentrations of ethanol. Antigen retrieval were performed using 1 mM EDTA unmasking solution (ZSGB-BIO, ZLI-9069) by microwave heating. After cool down, sections were incubated in 3% hydrogen peroxide for 30 min in dark at room temperature, blocked with goat serum (ZSGB-BIO, ZLI-9056) for 1 h at room temperature, and probed with corresponding primary antibody at 4 °C overnight. Afterward, the sections were incubated with biotinylated goat anti-rabbit/mouse IgG (ZSGB-BIO, PV-6000) for 15 min at room temperature. Immunostaining was visualized with diaminobenzidine (DAB) (ZSGB-BIO, ZLI-9018), and sections were then counterstained with hematoxylin (Servicebio, G1004) and mounted with cover slip using neutral resins.