Polyoxymethylene

CCRF-CEM and Ramos cells were cultured for 12 h in six-well dishes at a density of 3 × 10⁵ cells per well. Cells were washed three times with PBS and incubated with Sgc8-FSNPs, FSNPs, or FITC-Sgc8, respectively, in binding buffer on ice for 30 min in the dark. Cells were washed three times with PBS, fixed with 4 % polyoxymethylene (Sigma-Aldrich) for 20 min, and stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Co., USA) for 5 min in the dark. Finally, cells were washed three times with PBS and examined by fluorescence microscopy (Nikon DS-Ri1, Japan).

Biocoat matrigel invasion chambers (BD Biosciences, Bedford, MA, United States) were used to compare the effect of SLPI knockdown on in vitro invasion of AsPC-1, BxPC-3 and PANC-1 cells as previously described [20 (link),21 (link)]. Briefly, for the invasion assay, Costar Tran-swell 8 μm inserts were coated with 50 μg reduced serum Matrigel (BD Biosciences, Bedford, MA, United States). Invasion Chambers (BD China, Shanghai, China) were coated with Matrigel, and 1 × 10⁶ cells were added per chamber. Medium supplemented with 10% FBS was used in the lower chamber. Following incubation cells that had invaded through the membrane were fixed and stained before the membrane was removed and mounted on a slide for microscopic assessment. Invasive cells were visualized at x40 magnification and the number of cells in five random fields was counted and an average calculated.
For migration assays, the same procedure was used excluding the Matrigel. After 12 h, non-invading cells and media were removed, and cells on the lower surface of the membrane were fixed with polyoxymethylene (Sigma) and stained with 0.1% crystal violet (Sigma) for 0.5 h. Stained cells were counted under a microscope in five randomly selected fields, and the average was used to indicate cell migration and invasion. All experiments were performed in triplicate [21 (link)].

DPCs were grown in osteogenic medium containing 10 μM E3330. At days 3 and 5, ALP activity of E3330 treated and untreated DPCs was determined according to the manufacturer’s recommendations with an ALP kit (Jiancheng, Nanjing, China) and normalized on the basis of equivalent protein concentrations. The absorbance of each well was determined using a microplate reader at 520 nm. Calcium deposition in the extracellular matrix was determined by Alizarin Red S staining after 14 days of osteogenic differentiation. Cells were fixed in 4% polyoxymethylene (Sigma-Aldrich, USA) and then incubated in 0.1% Alizarin Red S solution (pH = 4.3; Sigma-Aldrich, USA). Calcium deposition was observed and visualized under a light microscope, and the picture of 6-wells plate was captured by camera.

To induce BM-MSCs into osteoblasts, cells were cultured in osteogenic differentiation medium (DMEM (low glucose) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 × 10⁻⁴ M L-ascorbic acid, 10⁻⁷ M dexamethasone, and 10⁻² M β-glycerophosphate disodium salt hydrate (Sigma-Aldrich)) for 14 days [17 (link), 18 (link)]. The differentiation medium was changed every 3 days.
Calcium deposition in the extracellular matrix was determined by Alizarin Red S staining after 7 and 14 days of osteogenic differentiation. Cells were fixed in 4% polyoxymethylene (Sigma-Aldrich) and then incubated in 1% Alizarin Red S solution (pH = 4.3; Sigma-Aldrich). Images of calcium deposition were captured with an Olympus IX51 microscope. To quantify the calcium deposition, 200 μL/well of 1% hydrochloric acid (Sigma-Aldrich) was added and absorbance was measured at 420 nm using a microplate spectrophotometer (BioTek).

Transwell invasion test was performed adopting a transwell chamber (Keygen, Nanjing, China) involving matrix gel with 8 μm pore size. The cells are digested and counted. Placing of 1 × 10⁶ cells was in the upper chamber with 100 μL FBS free medium, and the lower chamber was covered with 500 μL medium containing 10% FBS as a chemical attractant. After incubation in a wet incubator for 24 h, removal of non-migrating cells was on the upper membrane surface, fixation of the cells on the lower membrane surface was with 4% polyoxymethylene (Sigma, MO, USA), and staining was with 0.1% crystal violet (Sigma, MO, USA). The cells were counted and the images were recorded via shooting five random fields at 400 × magnification under a microscope (BX53, Olympus, Tokyo, Japan).