Phosphate buffered saline (pbs)

LBH589 (Selleck, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China), and diluted subsequently in DMEM or phosphate-buffered saline (PBS; Meilunbio, Dalian, China) to different working concentrations for use. To prevent the toxic effects, the final concentration of DMSO should not exceed 0.01% in all experiments.

Approximately 1 × 10⁶ T241-vector or T241-FGF-2 tumor cells in 50 μL PBS (catalog MA0015, Meilunbio) were s.c. implanted into each C57BL/6 mouse. A total of 1 × 10⁶ 5-8F shScrambled or 5-8F shFGF2 tumor cells in 50 μL PBS were s.c. implanted into each BALB/c-nude mice. For 4T1 orthotopic tumor models, 1 × 10⁶ 4T1-vector or 4T1–FGF-2 tumor cells in 50 μL PBS mixed with 50 μL Matrigel Matrix (catalog 354234, Corning) were injected into the mammary fat pad of female C.FVB-Tg(Cspg4-TK*)1Rkl/J mice. Tumor sizes were measured every other day with a calliper, and tumor volumes were calculated according to a standard formula: Tumor volume = length × width² × 0.52 (11 (link)). Tumor removal was surgically performed under anesthesia, when primary tumor volumes reached the size of 2.0–2.5 cm³. Mice were kept for an additional 4–6 weeks for metastasis detection. GFP⁺ metastatic nodules were detected by an IVIS system (VISQUE Invivo Elite, VIEWORKS). Lung tissues were subsequently paraffin embedded, stained with H&E, and examined under light microscopy.

Venous blood was drawn from this patient, his mother and three healthy volunteers. An equal volume of phosphate buffered saline (PBS) (Meilunbio, Dalian, China) was added to ethylene diamine tetraacetic acid (EDTA) anticoagulated blood. The diluted blood was carefully added to the top of Ficoll-Paque Plus media (Amersham Pharmacia Biotech, Baie-D'Urfé, Quebec, Canada) in a centrifuge tube (Corning, NY, United States) and centrifuged at 2,000 rpm for 20 min at room temperature. After being washed twice in PBS, the peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640 (Sigma‒Aldrich, United States) supplemented with 10% fetal bovine serum (FBS) (Sigma‒Aldrich, United States) at a density of 1×10⁶ cells/ml. Ultrapure Escherichia coli lipopolysaccharide (LPS) (Sigma‒Aldrich, United States) was added to the cell culture at a final concentration of 100 ng/ml for stimulation. After incubation in a 12-well plate at 37°C in 5% CO₂ for 24 h, the cells were harvested for subsequent experiments.

HaCaT cells and JB6 cells (5 × 10⁵ cells/well) were seeded in a six-well plate for the wound healing assay. When the cells were cultured to 90% confluence, the cells were pretreated with a low dose of mitomycin c (5 μg/mL) for 2 h. After removing the mitomycin c-containing medium and washing it once with PBS, a straight line was drawn vertically with a 200 uL pipette tipper well. Dropped cells were washed out with phosphate-buffered saline (PBS, Meilunbio, Dalian, China). And then, DMEM containing different concentrations of LQ (10, 20, 40 μg/mL) was added to each well. Groups of scratched areas were photographed at 0 h and 24 h using a microscope (Olympus, Tokyo, Japan). The scratch wound closure rate was determined by the following equation: Scratch wound closure rate = (Scratch area at 0 h − Scratch area at 24 h)/Scratch area at 0 h × 100%.

Cell viability was also determined by crystal violet staining as previously described^{31 (link)}. In brief, HEI-OC1 cells were seeded into a 24-well cell culture plate. After the designated treatment, the cells were washed in ice-cold phosphate-buffered saline (PBS; Meilunbio, MA0015) and fixed in cooled methanol on ice for 10 min. The cells were then stained with crystal violet solution (0.5% crystal violet in 25% methanol) for 10 min at room temperature. The cells were washed with tap water and allowed to dry. Stained cells were then extracted with 1% sodium dodecyl sulfate. After solubilization, the 100 μl per well dye extracts were measured at 570 nm using a microplate reader. The average OD value in the control cells was taken as 100% viability.