Vcam 1

HUVEC cells (1.0 × 10⁵ cells/well) were seeded in an 8-well slide chamber for 24 h and starved overnight, followed by pre-treatment for 1 h with IL-32θA94V (100 ng/mL) and a 6 h treatment with TNF-α (10 ng/mL). After the treatment, cells were fixed using 4% paraformaldehyde for 10 min followed by incubation with ice-cold methanol and blocking using 1% BSA in PBS for 1 h at 25°C. The cells were then incubated with primary antibodies against ICAM-1 and VCAM-1 (Beijing Solarbio Science & Technology Co., Beijing, China) overnight at 4°C, and with secondary antibodies conjugated to Cy3 (Merck Millipore, Darmstadt, Germany) for 1 h at 25°C, followed by DAPI staining. Two washing steps with PBS were performed between each step. Thereafter, cells were mounted in mounting buffer (Sigma-Aldrich) and examined under a fluorescence microscope.

The secretions of proteins were determined using ELISA assays. Following ELISA kits from Solarbio life sciences were used in this study: MMP-2 (Cat#SEKH-0253), MMP-9 (Cat#SEKH-0257), VCAM-1 (Cat#SEKH-0055), ICAM-1(Cat#SEKH-0053), COL1α1 (Cat#EH0958), and TGF-β1 (Cat#EH0287). The ELISA was performed according to the protocol from the manufacturer. The expression level was measured at the wavelength of 490 nm using a microplate reader.