Slgv004sl

An in vitro invasion assay was performed in triplicate using Growth Factor Reduced BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. Briefly, 3 × 10⁴ cells were seeded in the upper chamber with conditioned medium from PrSC treated with or without 100ng ml^-1 rFABP4, and the presence or absence of 10  μg ml^-1 of IL-8 blocking antibody, IL-6 blocking antibody, control goat IgG, or control mouse IgG (R&D Systems, Minneapolis, MN, USA). In the siRNA experiments, cells were treated with 50 nM FABP4 siRNAs for 24 hours before being seeded in the chambers. Subsequently, 20% FBS DMEM was placed in the lower chamber, followed by incubation for 24 hours. In some experiments, 1 × 10⁴ PrSC were seeded in the lower chamber with optimal medium. Sera from mice were collected, clarified by filtration (SLGV004SL; Millipore, Billerica, MA, USA), and used for ex vivo cell invasion assays. Briefly, 3 × 10⁴ cells were seeded in the upper chamber with medium containing 5% FBS or 5% mouse serum with or without 10  μg ml^-1 of IL-8 blocking antibody or 30 μM of BMS309403. DMEM with 20% FBS was placed in the lower chamber. Then, the non-invading cells in the upper chamber were removed and the membranes were stained with a Diff-Quik cell-staining kit (Sysmex, Kobe, Japan) to count the invading cells. The experiments were performed twice each in triplicate.

Culture supernatants were clarified by filtering through 0.22-μm filter (SLGV004SL; Millipore) and stored at −80°C until use. BM fluids were prepared as previously described (Kang et al., 2020 (link)) and stored at −80°C until use. For ELISA measurements, 50 µl of culture supernatants were analyzed with IL-6 (50-172-18; eBioscience) or TNFα (88-7324-22; eBioscience) ELISA kits according to the manufacturer’s instructions. For Luminex cytokine multiplex bead arrays, 25 µl of culture supernatants were analyzed for a custom-made panel of five cytokines (IL-1β, IL-6, GM-CSF, TNFα, MIP1α; Invitrogen, PPX-05) according to the manufacturer’s instructions. For Raybiotech 200 mouse cytokine arrays, 500 µl of pooled culture supernatants or BM fluids were sent per sample to Raybiotech for quantitative proteomics services using Mouse Cytokine Array Q4000 kit. Quantile normalization was performed for direct comparison of secreted cytokine profiles from different array experiments using R Bioconductor package, and hierarchical clustering was conducted using pheatmap package of R (https://cran.r-project.org/web/packages/pheatmap/index.html). The statistical significance of the different distributions of secreted cytokine under diverse biological conditions was determined using Kruskal-Willis test (P value <0.05, R version 3.3: https://www.r-project.org).