Lactate dehydrogenase ldh assay

MG-63 cells were incubated with anti-TPD52 antiserum (5 μg, 10 μg, 25 μg, 50 μg, and 100 μg) for 48 h. Since etoposide is a commonly used anti-osteosarcoma drug in clinical practice [30 (link)], it was selected as the positive control and used at a concentration of 50 and 100 μM. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8, Trans, Beijing, China) assay at 450 nm wavelength. The cell proliferation rate was calculated as (OD treatment − OD blank)/(OD control − OD blank) × 100%.
Anti-TPD52 antiserum-treated MG-63 cell culture supernatant was collected, and the cytotoxicity was determined by lactate dehydrogenase (LDH) assay (Beyotime, Shanghai, China) according to the manufacturer's protocol. The percentage of LDH release was calculated as (LDH treatment − LDH control)/LDH total lysis − LDH control) × 100%.

NaHS was administered instead of H₂S. NaHS and the Pyk2 inhibitor (PF431396) were obtained from Sigma-Aldrich; The FAK specific inhibitor (PF573228), the Src inhibitor (PP2), and the Rac1 inhibitor (NSC23766) were purchased from Selleck Chemicals. CES-siRNA (sc-142618) was obtained from Santa Cruz. Rhodamine was obtain from Cytoskeleton, and DAPI from Beyotime; Following antibodies were used: anti-Rac, anti-Fak, anti-p-FAK³⁹⁷, anti-p-FAK⁹²⁵, anti-Src, anti-p-Src, anti-β-actin, anti-Integrin β1, anti-Integrin β3, anti-Cavenolin-1,anti-p-Pyk2 (Cell Signaling); anti-CSE (Santa Cruz); anti-Integrin β1-FITC, anti-Galectin-3 (eBbioscience); anti-CD68 (Biolegend); Lactate dehydrogenase (LDH) assay from BeyotimeInstitute of Biotechnology.