Thp 1 cells

HEK293F cells were a gift from Nicola Burgess-Brown (University of Oxford), and THP-1 cells were from Sigma. Cell lines were confirmed mycoplasma free by a kit (MycoAlert™, Lonza) and by DAPI staining and authenticated functionally by protein production for HEK293F, and chemokine – induced migration for THP-1.

HK-2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured as described previously [25 (link)]. After serum starvation of HK-2 cells with DMEM containing 0.1% FBS or conditioned medium from MSCs for 24 h, 10 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA) was added to the cells directly. After 30 min (to investigate protein levels of p-Smad2) or 24 h (to investigate protein levels of α-SMA), HK-2 cells were collected and subjected to in vitro experiments.
THP-1 cells were also obtained from the American Type Culture Collection and cultured as described previously [25 (link)]. To induce differentiation of THP-1 cells into M1 macrophages, THP-1 cells were treated with 160 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 48 h. Then, the medium was replaced with conditioned medium from MSCs. After 24 h, the cells were collected and subjected to in vitro experiments.

THP-1 cells (human macrophage cell line) were purchased from the Duke core facility (ATCC # TIP202). Cells were cultured in high glucose (4.5 g/L) RPMI 1640 Medium containing 10% (v/v) FBS at 37°C. THP-1 cells were treated in RPMI media containing 250 nM PMA (Sigma) for 48 hours for differentiation and then washed with PBS to eliminate non-adherent cells. The adherent cells were differentiated (mature) human macrophages. To examine IL-23 and IL-17A secretion in THP1 cells, we purchased hIL-23 ELISA kit from Biolegend (# 437607, San Diego, CA) and hIL-17A ELISA kit from Meso Scale Diagnostics (# K151RFD-1, Rockville, MD). After seeding matured THP1 cells (500,000, 400 μl), we treated these macrophages with β-estradiol (1 ng/ml), paclitaxel (1 µg/ml), and their combination for 24 hours at 37°C. We collected 200 μl of the culture media for ELISA assays. We measured secreted cytokine levels using a Bio-Rad plate reader for hIL-23 and a MESO QuickPlex SQ 120 for hIL-17A, according to the manufacturer’s protocol.

THP-1 cells (ATCC, USA) were cultured in RPMI1640 medium containing 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37°C. Macrophage-like cells were differentiated from parental THP-1 cells by induction with phorbol-12-myristate-13-acetate (PMA, 200 nmol/L; Sigma, USA) for 24 h and were then infected with TIPE2 adenovirus and siTIPE2 or treated with culture medium containing glucose (25, 35, and 50 mmol/L) for 24 h.

THP-1 cells were purchased from Sigma-Aldrich, NZ (Cat. # 88081201) and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO₂. THP-1 cells, at a density of 0.5 × 10⁶ cells / well in 6-well plates, were differentiated into macrophages by treatment with 50 nM PMA for 72 h. Differentiated THP-1 cells were then treated with curcumin and CGA for 1 h, both individually and in combination, at a variety of concentrations (1–25 µM) as indicated in the figure legends. The cells, in the presence of bioactive compounds, were then incubated with LPS (100 ng/mL) for another 4 h to induce an inflammatory response. Cells were pre-treated with curcumin and CGA to ensure full uptake and equilibration prior to LPS stimulation, helping to rule out effects, due to kinetic differences in their absorption versus LPS action. The cell culture supernatants were collected and stored at −80 °C until used for TNF-α quantification by ELISA. The remaining cell monolayer was immediately used to assess cytotoxicity via MTT assay or extracted for subsequent qRT-PCR analysis.