Alizarin red solution

The RAW 264.7 cell line was purchased from American Type Culture Collection (ATCC® TIB-71™). C-PC was bought from Sigma-Aldrich (St. Louis, MO), dissolved in sterile water and stored at 4 °C. MTT assay kit, Alkaline phosphatase staining kit, and GAPDH antibody were also purchased from Sigma-Aldrich (St. Louis, MO). The following antibodies were bought from the company indicated in parenthesis: NFATc1 (SC-16657; Santa Cruz Biotechnology; Santa Cruz, CA), TRAP and CTSK (ab191406, ab19027; Abcam; Cambridge, United Kingdome), β3 Integrin, Caspase-3, caspase-9, c-Fos, and IκB-α (4702, 9662, 9504 S, 4384 S, 4814 S; Cell Signaling Technology; Danvers, MA), and HRP conjugated (mouse or rabbit) secondary antibodies (Santa Cruz Biotechnology; Santa Cruz, CA). Protein estimation reagents, molecular weight standards for proteins, and PAGE reagents were bought from Bio-Rad. Alizarin red solution was bought from Life-line Cell Technology (CM-0058; Fredrick, MD). Super Signal™ West Pico Chemiluminescent substrate was bought from Thermo Fisher Scientific (Waltham, Massachusetts). Rhodamine phalloidin and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Antibody to L-plastin (SC-16657; Goat) was bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to GAPDH was purchased from R & D Systems (Minneapolis, MN) and Sigma (St. Louis, MO). Protein estimation reagent, molecular weight standards for proteins, and PAGE reagents were bought from Bio-Rad. Cy2- and Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). HRP-conjugated secondary antibodies for immunoblotting were obtained from GE Healthcare. Antibody to phosphoserine (p-Serine) was bought from Zymed laboratories (61–8100) or Millipore (AB1603). Alizarin red solution was bought from Life-line Cell Technology (CM-0058; Fredrick, MD) Rhodamine-phalloidin and other chemicals were purchased from Sigma (St. Louis, MO).

Alizarin red staining was used to visualize and quantify in vitro calcification. The cells were washed with 1 × PBS, fixed with 10% formaldehyde for 15 min, washed twice with distilled water, incubated with 2% Alizarin Red solution (LifeLine Cell Technology) for 20 min at room temperature and finally washed three times with distilled water to remove excess stain. Then 100 mM hexadecylpyridinium chloride (Sigma-Aldrich) was added and incubated for three hours at room temperature before absorbance was measured at 540 nm (Infinite 200Pro, Tecan i-control) to quantify the Alizarin red staining.