Ab9797

Migration assays were carried out in a 24-well transwell system equipped with porous (8 μm) polycarbonate membranes. 2 × 10⁵ MB cells were resuspended in 600 μL of growth medium supplemented with 1% FBS and plated into the lower chamber of the transwell system; 5.0 × 10⁴ BMSC cells were resuspended in 200 mkl of growth medium supplemented with 1% FBS and plated into Transwell inserts which were then placed into another transwell system with the lower chamber filled with 600 μl of serum-free medium. After 24 hours the cells on the inserts or in lower chambers were transfected as indicated, 6 hours after the transfection the media was changed and the inserts with BMSC, were placed into the wells with MB cells and incubated at 37 °C for 24 h. When indicated, CXCR4 (Abcam, #ab10403) or SDF1 (CXCL12) (Abcam, #ab9797) antibodies were added to the medium at 10 mkg/ml and 4 mkg/ml concentrations respectively. The inserts were then discarded, and upper sides of the filters were swabbed to remove the cells that did not cross the membrane. The cells present on the lower side of the filters were then fixed in 4 % paraformaldehyde, stained with DAPI and the counted under the microscope. All the experiments were performed in duplicate. The following control antibodies were used: rabbit IgG control (AB-105-C, R&D systems) and mouse IgG2b isotype control (MAB004, R&D systems).

Immunohistochemical staining was carried out as described previously.¹⁶ A rabbit anti‐SDF1 (CXCL12) polyclonal Ab (1:200 dilution, ab9797; Abcam), mouse anti‐α‐smooth muscle antigen (α‐SMA) monoclonal Ab (1:50 dilution, M0851; CiteAb) and mouse anti‐CD8 monoclonal Ab (prediluted, clone C8/144B; DAKO) were used. We evaluated the correlation between CXCL12 expression and OSCC histological grades based on the World Health Organization (WHO) and YK classifications.¹⁷, ¹⁸ Immunohistochemistry was performed and evaluated by pathologists (Akira Takasawa and Makoto Osanai). To accurately quantitate CXCL12 expression levels over entire tumor areas, we measured the positively stained areas using ImageJ software (NIH) as described previously.¹⁹ Muscle cell areas were quantified using ImageJ software.

Recombinant human IL-6 (#AF-200-06, PeproTech, Rocky Hill, USA), recombinant murine SDF-1a (CXCL12) (#250-20A, PeproTech, Rocky Hill, USA), recombinant murine MCP-1 (#250-10, PeproTech, Rocky Hill, USA), Anti-hIL-6-IgG (#mabg-hil6-3, InvitroGen, Carlsbad, USA), Anti-SDF-1 (CXCL12) antibody (ab9797, Abcam, Cambridge, UK), Anti-MCP-1 antibody (ab25124, Abcam, Cambridge, UK).

TRAP staining (Sigma-Aldrich, Missouri, USA) and immunostaining were performed according to the manufacturer’s instructions. Serial sections were incubated with primary antibodies to anti-Osterix (Abcam, ab22552; Santa Cruz, sc-393060), anti-Ki-67 (Immunoway, YM-3064), anti-p-S6 ribosomal protein (Ser235/236) (Cell Signaling Techonlogy, #2211), anti-Cxcl12 (Abcam, ab9797), anti-CXCR4 (Abcam, ab124824), anti-collagen X (Abcam, ab58632), and anti-MMP-13 (Abcam, ab39012) overnight at 4 °C. For immunofluorescent staining, secondary antibodies conjugated with fluorescent tags were added and slides were incubated at room temperature for 1h in dark. Photomicrographs of sections were captured to perform histomorphometric measurements on the entire area of the tibial subchondral bone with ZEISS Scope A1 (Zeiss, Heidelberg, Germany), Quantitative histomorphometric analysis was conducted in a blinded fashion using the OsteoMeasure XP Software (OsteoMetrics, Inc., Atlanta, USA). The number of positively stained cells in the entire tibia subchondral bone area was counted in three sequential sections per mouse in each group.

The mouse-anti-human monoclonal antibody R6G9 (IgG_2a), which is directed against the extracellular domain of human integrin subunit β6, was obtained from Chemicon International (Temecula, CA, U.S.A.). The following monoclonal antibodies were obtained from Abcam (Cambridge, MA, U.S.A.): ab5694 and ab28244, which target the CAF markers α-SMA and FAP, respectively: ab27969, which targets transforming growth factor β (TGF-β); and ab9797, ab9695, ab16828, and ab6672, which target the four cytokines stromal cell-derived factor-1 (SDF-1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and interleukin 6 (IL-6). EP1186Y and EP1254 monoclonal antibodies, which target matrix metalloproteinase (MMP) 3 (MMP-3) and MMP-9, respectively, were also purchased from Abcam. Reagents for SDS/PAGE and molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.). The C–X–C chemokine receptor type 4 axis (CXCR4) antagonist AMD3100 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).