Af1433

Cancer cells were cultured in a 10-cm dish to 80% confluency. The cells were washed with PBS and then incubated with FBS-free medium. After 48 hours of incubation, the medium was collected, centrifuged (2000 rpm, 10 minutes) and filtered using a 0.2-μm filter to obtain cancer cell conditioned medium (Ca.-CM). The CM was used without additional FBS. CAF-derived cancer [CAF (Ca.)] and TAM-derived cancer media were prepared by adding Ca.-CM to HSCs or M0 macrophages for 48 hours (Figure 1A). Then, the medium was exchanged once, and the supernatant was collected. These supernatants were used as CAF conditioned medium (CAF-CM) and TAM conditioned medium (TAM-CM). CAFs derived from TAM-CM [CAFs (TAM)] and CAF-CM [CAFs (CAF)] were prepared in the same way (Figure 1C). To neutralize OPN in the CM, an OPN antibody (AF1433; R&D Systems, Inc., MN, USA) was added at a concentration of 1.0 μg/mL. In the examination of adding OPN, OPN (1433-OP; R&D Systems, Inc., MN, USA) was added to HSCs at concentrations of 0.1, 0.5 and 1.0 μg/mL, the medium was exchanged once in the same manner as above, and the CM was collected.

To determine the expression of osteogenic markers, $3\times {10}^4$ passage 5 dental pulp cells were seeded onto 24 mm diameter nanofiber-PDMS scaffolds in 24-well plates (n = 5), and other procedures, such as scaffold sterilization and fixation, were performed as described above.
After fixation and washing three times with 0.01% Tween 20-PBS solution (PBST), 1% Triton X solution was added to each well for 15 min to permeabilize. After washing three times with PBST, 1% bovine serum albumin (BSA) solution was added to each well and incubated at room temperature for 1 h for blocking. Appropriate concentrations of primary antibody (polyclonal anti-human osteopontin goat IgG, R&D Systems AF1433), secondary antibody (anti-goat IgG fluorescein isothiocyanate (FITC)-conjugated, tetramethylrhodamine (TRITC)-conjugated phalloidin), Alexa Fluor^® 594 and DAPI solution were continuously added for 1 h, except 5 min for DAPI. Each sample was washed three times with PBST between and after treatment. All scaffolds were mounted on a cover glass after staining was performed and imaged with a high-resolution fluorescence microscope (Nicon N-Storm, Tokyo, Japan).
The intensity of each image was analyzed with the ROI manager tool in ImageJ, randomly selecting five 50,000 pixel-sized square image sites.