Anti cd44 antibody

Western blotting and Immunohistochemically staining were performed as previously described [35 (link)]. The primary antibodies used in WB concludes of ESR1 (ab108398, 1:2000, Abcam), cyclin d1(#2978, 1:2000, Cell Signaling Technology), DICER1 (#3363, 1:2000, Cell Signaling Technology), NICD (#2421, 1:1000, Cell Signaling Technology), Vinculin (#13901, 1:5000, Cell Signaling Technology), NUMB (ab14140, 1:2000, Abcam), GADPH (#5174, 1:5000, Cell Signaling Technology). For immunobiological assay, the slides were incubated with anti-CD44 antibody (#3570, 1:500, Cell Signaling Technology), ALDH1 antibody (#54135, 1:500, Cell Signaling Technology) and NUMB antibody (ab14140, 1:1000, Abcam).

To evaluate tumorigenesis, 10⁴, 10³, or 10² untreated or DKG pre-treated cells were mixed with Matrigel (1:1, Corning) and injected subcutaneously in the left and right flanks of the same NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mouse, respectively. Tumor weight was measured 2 months after injection. To further investigate the role of HIF-1α in the xenograft model, 10⁴ or 10⁶ of MDA-MB-231/shCON or shHIF-1α cells pre-treated with DKG were resuspended in 100 μl serum-free DMEM media, and then injected subcutaneously into the right flanks of six-week-old female NSG mice. The tumor volumes were measured twice weekly and the mice were euthanized on day 65, and the tumors were harvested and weighed. The tissue sectioning and the IHC staining were done by the Pathology Core at City of Hope, using an anti-CAIX antibody (Genetex), anti-CD44 antibody (Cell Signaling) and anti-CD133 antibody (Fitzgerald). Isolation and culture of tumor cells from the mouse xenografts were performed as previously described [78 (link)]. Briefly, the tumors were minced into small pieces and then digested in collagenase-hyaluronidase-DNase I (Sigma) buffer for 60 minutes at 37°C. The cell suspension was then passed through a 100 μm cell strainer (BD Biosciences) and the resulting cells were plated on petri dishes and maintained in the growth medium for culturing MDA-MB-231 cells.

Monolayers of hDPSCs were cultured and treated with HA-based gels in standard and osteogenic media in four-well covered glass chamber slides. After 48 h of treatment, the cells were fixed with 4% w/v paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized in 0.2% Triton X-100 v/v in PBS for 1 h. Non-specific sites were blocked using blocking buffer solution (PBS containing 10% v/v bovine serum and 1% w/v BSA). The cells were then incubated with anti-CD44 antibody (Cell signaling, Leiden, The Netherlands, 156-3C11) overnight at 4 °C followed by incubation with corresponding secondary antibody for 2 h at room temperature. Nuclei were stained with 20-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2.50-bi-1H-benzimidazole trihydrochloride hydrate, bisBenzimide (Hoechst 33342, Milan, Italy) and actin filaments were stained using phalloidin tetramethylrhodamine B isothiocyanate (Sigma-Aldrich, Milan, Italy, 51927). Fluorescence images were captured using a fluorescence microscopy system (Nikon, Tokyo, Japan).