Recombinant human ccl2

Cells were cultured in Collagen: Matrigel, using procedures adapted from previous studies (Berens et al., 2015 (link)). Rat tail collagen (3.5 mg/ml) (Corning, cat. no. 354236) was mixed at a 4:1 ratio with setting solution comprised of: 1× EBSS, 75 mM NaOH and 290 mM NaHCO₃. The collagen solution was then diluted 1:1 with Growth Factor Reduced Matrigel (BD Pharmingen, cat. no. 354230). 96-well plates were coated with 40 µl/well of the Matrigel: Collagen solution for 30 min at 37°C. Breast cancer cells (2500) were re-suspended in 200 µl growth medium containing 2.5% Matrigel and seeded in each well for 24 h. Cells were treated with or without recombinant human CCL2 (Peprotech, cat. no. 300-04) at 60 ng/ml or 100 ng/ml. Cells were cultured for up to 10 days, with the medium changed at day 4 and 8. Images were captured every 2 days using the EVOS FL Auto Imaging System (Invitrogen) at 10× magnification, four fields per well.

Oro-A was a gift from Dr. Tony Jer-Fu Lee and obtained from MedChem Express (Monmouth Junction, NJ, USA) . Extraction and purification of Oro-A was described previously [57 (link)]. Oro-A was dissolved in 100% dimethyl sulfoxide (DMSO) as a 100 mM stock solution. Recombinant Human CCL2 was obtained from PeproTech (Rocky Hill, NJ, USA).

THP-1 cells were cultured in RPMI medium 1640 + GlutaMAX from Gibco supplemented with 8% fetal calf serum (FCS), 10 mM HEPES, 1 mM sodium pyruvate (sigma) and streptomycin (50 μg/mL) in a humidified atmosphere of 5% CO₂ at 37 °C. THP-1 cell migration was studied in a modified Boyden chamber assay using a transwell chamber system (Fluoroblock, 8 µM pore size, BD Bioscience, Heidelberg, Germany). 2.5 × 10⁴ cells were seeded in the insert in RPMI medium with 0.1% BSA. Migration towards the stimulus or supernatant derived from HAoSMC conditioned media which was added to the lower chamber, was determined after 14 h. For conditioned media HAoSMC were treated with 100 nM AEA (2 h) and 10 ng/mL IL-1β (3 h). For further analysis supernatant was incubated with 50 ng/mL recombinant human CCL2 (#300-04, Peprotech) or 1:1000 diluted CCL2 antibody (#sc-52877, Santa Cruz).

The human prostate cancer cell lines (DU145 cells and PC-3 cells), human acute monocytic leukaemia cell line (THP-1 cells), IMR-9 cells (human normal lung fibroblast) were obtained from the American Type Culture Collection. The DU145 cells and PC-3 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA), supplemented with 10% FBS at 37 °C and 5% CO₂. The THP1 cells were cultured in RPMI-1640 medium (RPMI) (Invitrogen Corp., Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. IMR-90 cells were cultured in Minimum essential medium Eagle (Invitrogen Corp., Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Recombinant Human CCL2 was obtained from Peprotech. Dihydroisotanshinone I (DT) was obtained from ChemFaces Natural Products Co., Ltd., China (Catalog number: CFN-90162, the purity of dihydroisotanshinone is 98% and its solubility in DMSO is > 5 mg/mL). Human prostate cancer cells and macrophages were cultured to 60–70% confluence prior to treatment. Medium was then replaced with fresh medium containing dihydroisotanshinone in DMSO (dimethyl sulfoxide) at the indicated concentrations. Cells treated with DMSO alone were used as untreated controls.

Human monocytes were treated with demeclocycline (10 μM). After 1 h of incubation at 37°C with 5% CO₂, IFNγ (100 ng/ml)/IL-1β (100 ng/ml) was added. After 24 h, human monocytes were harvested and resuspended in RPMI 1640 media supplemented with 2% penicillin/streptomycin, 10% fetal bovine serum, L-glutamine, and 1 mM sodium pyruvate. Two hundred thousand cells were plated onto the filters of 5 μm pore size ChemoTx plates (NeuroProbe). Recombinant human CCL2 (Peprotech) (10 ng/ml) was diluted in supplemented RPMI 1640 media and 300 μl/well was added into wells below the filter so as to provide a chemotactic stimulus. Two controls were used in this assay. The first control was medium only. The second one was chemokinetic control where the cells plated onto the filter contained the 10 ng/ml of CCL2 as in the underlying well. To obtain a standard curve, halving numbers of cells were plated ranging from 0 to 200,000. Cells were incubated at 37°C in humidified air with 5% CO₂ for 16 h. They were then washed off the top of the filter and the plate spun at 1,400 rpm for 10 min at room temperature. One hundred and fifty microliter of the media was discarded in the microplate and replaced with 15 μl of alamarBlue® (Invitrogen). The plate was then placed at 37°C in humidified air with 5% CO₂ for 4 h and signal was read at 570 nm. This assay was also conducted with mouse BMDM.