Rpmi 1640

Transwell migration and invasion assays were conducted as previously described [13 (link)]. For invasion assay, the upper compartment of the transwell chamber (8 μm pore size, 24 wells, Millipore, Billerica, USA) were precoated with 10 μL of diluted Matrigel (Matrigel and RPMI 1640, 1:3, v/v, BD Biosciences, Sparks, USA). Cells were suspended in serum-free medium and seeded into upper chambers at a density of 3 × 10⁴ and 5 × 10⁴ cells for migration and invasion assays, respectively. To examine the effect of signaling agonists and inhibitors on the invasiveness of GC cells, the Matrigel-transwell system were cultured with or without IFNγ (50 ng/mL, R&D Systems, Minnesota, USA), STAT1 siRNA (40 nmol/L), JAK inhibitor I (15 ng/mL, Santa Cruz Biotechnology), ZEB1 siRNA (50 nmol/L), and IRF1 siRNA (50 nmol/L). RPMI-1640 medium containing 10% FBS and indicated inhibitors or agonists were added to lower chamber (600 μL/well). After incubation for 24 h, the non-invasive cells on the top surface of upper chamber were carefully removed with a cotton swab. Cells were fixed, stained and counted. Each experiment was repeated three times.

All biochemical assays were performed in triplicate on at least three independent occasions for the determination of mean values reported. All the reagents including foetal bovine serum (FBS) and cell culture growth medium (MEM, DMEM and RPMI-1640) were purchased from BD Biosciences. CA-4 was purchased from Sigma Aldrich.

All protocols involving animals were approved by the Animal Care and Use Committee of the Beijing Institute of Biotechnology and performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. To assess the tumor formation capacity, 3 × 10⁶ MC38 murine colon adenocarcinoma cells, with or without 3 × 10⁶ hLC-MSCs, were suspended in a 100-μL volume at a 1:1 ratio in either standard RPMI 1640 medium alone or medium with Matrigel matrix (BD Biosciences), and transplanted subcutaneously into 4- to 5-week-old C57BL/6N mice. Tumor formation was monitored weekly.

786-O, 769-P, A704, MDA-MB-231 and 293T cells were purchased from ATCC. These cells were not listed by the International Cell line Authentication Committee (ICLAC) as misidentified cell lines (3 October 2014). Renal carcinoma cell lines were cultured at 37 °C in RPMI 1640 (Invitrogen) supplemented with 10% FCS (HyClone). MDA-MB-231 cells were maintained in a 1:1 mixture of DMEM (Invitrogen) and RPMI 1640 supplemented with 10% FCS and 5% Nu-Serum (BD Biosciences), as described previously11 (link). 293T cells were cultured in DMEM supplemented with 10% FCS. 293FT cells were purchased from Invitrogen, and cultured at 37 °C according to the manufacturer's instructions. Plat-E cells were a gift from Dr Kitamura (Tokyo University), and were cultured at 37 °C in DMEM containing 10% FCS. All cell lines were determined to be free of mycoplasma using cytochemical staining of DNA with 4',6-diamidino-2-phenylindole (Sigma-Aldrich). No antibiotics were used in our cell cultures to avoid latent infection of mycoplasma to cultured cells.

Male non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice (4 weeks old) were purchased from the Shanghai Institute of Material Medicine of the Chinese Academy of Science and raised under specific pathogen-free conditions. Animal care and experimental protocols were conducted in accordance with guidelines established by the Shanghai Medical Experimental Animal Care Commission. Ethical approval was obtained from the research ethics committee of Zhongshan Hospital. HCCC9810-BAP1, HCCC9810-Mock, HCCC9810-BAP1C91A, RBE-shBAP1, and RBE-Mock cells (5 × 10⁶) were suspended in 200 μL serum-free RPMI-1640 and Matrigel (BD Biosciences; 1:1), and then injected subcutaneously into the flanks of NOD/SCID mice. Inhibition of ERK1/2 or JNK/c-Jun signaling pathways was performed by intraperitoneal injections of 25 μM/kg U0126 or 50 μM/kg SP600125 thrice a week from day 15 after inoculation, respectively. Inhibition of ERK1/2 or JNK/c-Jun signaling pathways was confirmed at the end of study using immunohistochemistry. The tumor volume was measured every 3 days with a caliper and calculated in mm³ using the following formula: V = length × width²/2. Upon euthanizing the mice, the tumors were removed, weighed, and photographed.

HL-60 cells were grown in RPMI 1640 (Biochrom) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin in a 5% CO₂ humidified atmosphere at 37°C. Cells (2 × 10⁶) were cultured in the presence of all-trans-retinoic-acid (ATRA; Sigma) in a final concentration of 1 μM for 5 days. Cell differentiation was assessed by apoptosis rate quantification by flow cytometry (FACSCalibur, BD).
In some experiments, freshly isolated human neutrophils, the Raji line of lymphoblast-like cells, HL-60, ATRA-differentiated HL-60 cells, Jurkat and THP1 cell lines were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 1% or 3% of serum, respectively.