Actin tracker red rhodamine

Tan IIA was purchased from Beijing Bethealth People Biomedical Technology (Beijing, China; purity ≥ 98%). Complete Freund's adjuvant (CFA) was purchased from Becton, Dickinson and Company (USA). Foetal bovine serum was obtained from Corning (USA), and penicillin/streptomycin was obtained from Thermo Fisher (USA). RANKL was obtained from Novoprotein (Suzhou). Cell fixative was obtained from Biorigin (Beijing). The Tartrate-Resistant Acid Phosphatase (TRAP) Stain Kit was obtained from Solarbio (Beijing). Actin-Tracker Red-Rhodamine was obtained from Beyotime Biotechnology (Beijing). A lactate dehydrogenase assay kit was obtained from Solarbio (Beijing). The NAD/NADH assay kit was obtained from BioXcellence (Beijing). Rabbit anti-mouse IgG was obtained from Abcam (Beijing). The desthiobiotin iodoacetamide (DBIA) probe was obtained from ChomiX Biotech (Nanjing). The following click chemistry reaction and LC‒MS/MS reagents were used in this study: TBTA (1770049), TCEP (C4706), rhodamine-N3 (83689) and CuSO4 (C1297) were purchased from Sigma (USA); TMT 10plex™ reagent (A34808), high-capacity neutravidin agarose resin (A53031) and sequencing grade modified trypsin (90057) were purchased from Thermo Fisher (USA).

Human ovarian cancer cell line A2780 were kindly gifted by the Cancer Research Institute, Zhejiang Cancer hospital (Hangzhou, China). Human ovarian cancer cell lines SKOV3 were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were authenticated by DNA(STR) profiling by iCell Bioscience Inc (Shanghai, China). SKOV3 was cultured with McCoy 5 A, HEK293T cell was cultured with DMEM, IOSE80 AND A2780 was cultured with RMPI 1640 medium. Human peritoneal mesothelial cells were isolated from the tumor-free omentum of patients without malignancy according to the methods described in another study [17 (link)]. Primary peritoneal mesothelial cells were cultured on collagen-coated plates with RPMI-1640 media. All media were supplemented with 1% penicillin-streptomycin and 10% FBS and incubated in 37 °C humidified atmosphere with 5% CO2. Cell Proliferation and Toxicity Detection Kit (CCK-8, CK101-01, DIB Data Inventory Biotechnology) were used according to manufacturer’s instruction. ADAM10 inhibitor GI254023X (S8660) was purchased from Selleck. F-actin was visualized with Actin-Tracker Red-Rhodamine (C2207S, Beyotime).

The following antibodies were used in this study: anti-GYS1 (ab40810, abcam), anti-PER2 (NB100-125, Novus Bio), anti-PER2 (sc377290, Santa Cruz), anti-NFκB p65 (sc-8008, Santa Cruz), anti-NFκB p65 (ab16502, abcam), and anti-Flag (#14793, CST). ELISA kit for mouse was purchased from eBioscience. Actin-Tracker Red-Rhodamine (C2207S) was purchased from Beyotime. Per2 shRNA lentiviral particles were purchased from Genepharma.

A Cell Counting Kit-8 (CCK-8) (M4839, AbMole, China) was used to assess the viability of the rBMSCs seeded in all groups. Briefly, control and experimental Ti surfaces were placed in 24-well plates and seeded with 1 ×10⁴ rBMSCs/mL DMEM. All the samples were cultured at 37 °C in 5% CO₂, and the media was changed every 3 days. After 1, 4, and 7 days, the specimens were removed from the 24-well plate and washed three times with phosphate-buffered saline (PBS) before being transferred to new 24-well plates. Then, 500 µl of media containing 10% CCK-8 solution was added to each well, which was followed by 2 h of incubation, after which the optical density (OD) of the media was read at 450 nm. After 3 days, the cells were stained with DAPI Staining Solution (Beyotime Biotechnology, China) and Actin-Tracker Red-Rhodamine (Beyotime Biotechnology, China) for cytoskeleton analysis via an inverted epifluorescence microscope (IX71, Olympus, Japan).