Rhodamine phalloidin

Cell proliferation was investigated using the CCK-8 assay. The seeding density of the cells was 1 × 10⁴/cm², and the detection time points were 1, 3 and 5 days. The other detailed procedures were nearly similar to the procedures of the cell attachment test. The modified OD values on days 3 and 5 were normalized to the OD values on day 1 because the numbers of attached cells on the different materials were different on day 1.
The cell morphology on the samples was observed via confocal laser scanning microscopy (CLSM). After the cells were cultured on the samples for 5 days, the cells were gently washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min and stained with rhodamine phalloidin (5 U/mL, Biotium, Hayward, CA, USA) for 30 min. The filamentous actin of the cell cytoskeleton was visualized using a CLSM (TCS SP2, Leica, Heidelberg, Germany).

Cells were seeded into Oris™ 96-well plate with silicon stoppers in place (CMA1.101). Cell density was optimized for each cell type, PC-3 and MDA-MB-231 were seeded at 2.5×10⁵ cells/mL or 6×10⁴ cells/mL. Stoppers were removed 12 hours post seeding according to the manufacturer's protocol and treatments were added, Taxol (5 nM) (Selleckchem), H10 peptide (10 μg/ml), scrambled peptide (10 μg/ml), mouse anti-AGR2 antibody (1:50 of 2 μg/ml) (Santa Cruz Biotechnology), mouse-IgG (Santa Cruz Biotechnology), DMSO as control. After 48 hours, growth medium was aspirated and cells were fixed with 4% paraformaldehyde (Alfa Aesar) for 15 minutes at RT and washed with PBS. Cells were stained with Rhodamine phalloidin (Biotium) 1:40 and Hoechst (Invitrogen) 1:10,000 for 20 minutes at RT and washed with PBS.

For the in vitro assay, scaffolds were washed with PBS, blocked with 3% bovine serum albumin (BSA) for 30 min, and incubated overnight at 4°C with primary antibodies against IGFBP3 (1:50, rabbit anti‐human, Santa Cruz) in addition to 2U of Rhodamine Phalloidin (Biotium, Hayward, CA); DAPI was used to stain nuclei.
For the in vivo assay, the scaffolds, retrieved at 7 days in vivo, were embedded in optimal cutting temperature compound, and snap frozen at −20°C. Sections (8 µm thick) were incubated overnight at 4°C with primary antibodies against Sca‐1 (1:500, 7 H4 L3, Invitrogen, CA) and PDGFR‐α (1:500, Invitrogen). Secondary antibody labeled with Alexa Fluor 488 (1:100, donkey anti‐rabbit) or Cy3 (1:100, goat anti‐rat, ZSGB‐BIO, Beijing, China) were used as appropriate. DAPI was used to stain nuclei. The number of Sca‐1⁺ PDGFR‐α⁺ cells were counted and the percentage of Sca‐1⁺ PDGFR‐α⁺ cells to the total cells were determined Fluorescence images were acquired using a Two Photon Laser Scanning System (LSM 510 NLO, Zeiss, Oberkochen, Germany). A total of three images per animal distributed within the defect area, with 800× magnification, were analyzed.

Anti-RSU-1 rabbit polyclonal antibody for immunoblotting was kindly provided by Dr. Mary Lou Cutler, Professor at the Uniformed Services University of the Health Sciences, Bethesda USA. Anti-pSTAT6 and anti-STAT6 were obtained from Cell Signaling. Anti-MMP13 was purchased from Abcam. Phospho-STAT6 inhibitor, AS1517499, was obtained from Axon Medchem. RSU-1 siRNA was purchased from Santa Cruz Biotechnology. Rhodamine-Phalloidin was obtained from Biotium and 4′,6-Diamidino-2-Phenylindole (DAPI) was obtained from Roche. Transwell inserts were purchased from Greiner Bio-One and Matrigel as well as Collagen I was obtained from Corning. QIAzol Lysis Reagent was purchased from QIAGEN.

The cell spreading was investigated by observing cells with SEM and detecting the filamentous actin of the cytoskeleton with a confocal laser scanning microscope (CLSM) after incubation for 24 h. To observe cells with the SEM, the MC3T3-E1 cells on the material surfaces were fixed with 2.5% glutaraldehyde for 15 min, dehydrated with gradient ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min, treated with hexamethyldisilazane (HMDS; Sigma-Aldrich, USA) for 10 min, sputter-coated with gold, and observed with SEM (S-4800, Hitachi, Japan). To observe the cytoskeleton with CLSM, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and stained with rhodamine phalloidin (5 U/mL; Biotium, USA) for 30 min. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, 0.1 μg/mL; Sigma-Aldrich, USA) for 10 min. The cytoskeletons and nuclei were visualized using a CLSM (Leica TCS SP2, Heidelberg, Germany).