Sc 13133

The TMA was constructed at the Department of Pathology in Lund in 2013. Duplicate 1 mm cores from formalin-fixed paraffin-embedded tissue blocks of representative tumor tissue were collected and assembled into a recipient TMA block using a semi-automated tissue array device (Beecher Instruments Inc., Sun Prairie, WI, USA). Sections of 4 μm were placed on glass slides and stored at −20 °C until immunohistochemical staining. Deparaffinization and antigen retrieval were performed using an automatic PT Link system (Agilent Technologies, Santa Clara, CA, USA). Immunohistochemistry was performed using the Autostainer Plus with the EnVision FLEX high-pH kit, according to the manufacturer’s instructions (Agilent Technologies). A previously well-validated mouse monoclonal D-6 antibody (sc-13133 Santa Cruz Biotechnology, CA, USA; diluted 1:750) was selected for the VDR staining due to its high sensitivity and specificity [21 (link)]. The dilution was optimized on a selection of tissue samples from different breast cancer subtypes to obtain a good separation between clearly negative results (blue) and strong intensity (brown) (Images in Figure 1 and Figure 2).

For western blotting, anti-VDR (D-6): sc-13133, anti-Fbxo32/MAFbx (F-9): sc-166806, anti-Trim63/MuRF1 (C-11): sc-398608 (Santa Cruz), and anti-β-Actin: 010-27841 (Wako) antibodies were used.

OSE cells (n = 3 patients) were plated at 100,000 cells/well in a 6-well plate. Twenty-four hours post plating, cells were treated with 0 (control) or 5 nM calcitriol (biologically active form of VD; Sigma-Aldrich) supplementation in the culture medium for 72 hrs. Cells were harvested for Western blot, as previously described [50 (link)]. Briefly, total protein was extracted using a RIPA buffer containing 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and 25 mM Tris-HCl (pH 7.6), electrophoresed on 4–12% Bis-Tris gel, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) nonfat milk in 10 mM Tris-buffered saline (pH 8.0), and then incubated with mouse anti-human VDR (1:250; sc-13133; Santa Cruz Biotechnology, Inc.) or α-Tubulin (loading control; 1:24,000; T6074; Sigma-Aldrich) antibody at 4°C overnight. The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, Burlingame, CA, USA). Images developed on the film were scanned using the Konica Minolta Bizhub C368 (Konica Minolta, Wayne, NJ, USA). Densitometry analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cartilage biopsies, two-day spheroids and three-week spheroids were fixed in 4% formalin overnight. Spheroids were embedded in 1% agarose before, along with the cartilage biopsies, they were cast and cut into 4 μm sections. Slides were rehydrated and antigen retrieval was achieved by placing the slides in a 60 °C citrate buffer for 60 min. The SuperPicture kit (Cat. no. 879673, Novex, LifeTechnologies) was used, along with specific antibodies targeting 1α-hydroxylase (Cat. no. ABIN2118284, Antibodies-online.com) and VDR (Cat. no. Sc-13,133, Santa Cruz) [15 (link)]. As previously described [16 ], slides from two-day and three-week controls and vitamin D-treated spheroids were prepared and stained with Alcian blue to detect glycosaminoglycan production. Pictures were developed using the Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany), and the images were evaluated by three investigators using the Bern score to assess chondrogenicity [17 (link)].

Cell protein homogenates (25 μg) were separated by electrophoresis in 12% polyacrylamide gels, transferred to nitrocellulose membranes, and blocked overnight with 5% nonfat dry milk. The membranes were washed and incubated in the presence of the following monoclonal antibodies: anti-IL-1R1, anti-IL-1R2, anti-TNFR1, anti-TNFR2, anti-VDR (sc-393998, sc-376247, sc-8436, sc-393614, and sc-13133, respectively; Santa Cruz Biotechnology, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, MAB374, Millipore, Milford, MA, USA) overnight at 4°C. Membranes were incubated in the presence of secondary antibody conjugated with horseradish peroxidase (sc-2031, Santa Cruz Biotechnology) for 2 hours at room temperature. The immunoblots were visualized by chemiluminescence using ECL Plus (Amersham Pharmacia, UK).

Total protein lysates were extracted from lung tissues or MLg2908 cells by homogenization in the Laemmli sample buffer. An equal amount of proteins (30 μg per lane) were separated by electrophoresis on 8% polyacrylamide gels, and the proteins were electro-transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) overnight. The membranes were then incubated with primary antibodies as follows: anti-β-actin (1:1000, Santa Cruz), anti-α-smooth muscle actin (SMA) (1:1000, CBL171, Millipore), anti-transforming growth factor (TGF)-β1 (1:1000, ab92486, Abcam), anti-collagen type 1 (Col I) (1:1000, ab21286, Abcam), anti-fibronectin (FN) (1:1000, F7387, Sigma-Aldrich), anti-vitamin D receptor (VDR) (1:1000, sc-13133, Santa Cruz), anti-renin (1:1000, sc-133145, Santa Cruz), anti-AT1R (1:1000, ab124734, Abcam) and anti-AGT (1:1000, sc-374511, Santa Cruz). Secondary antibody was horseradish peroxidase-conjugated anti-IgG (ZB-2301, ZB-2305, ZSGB-BIO). The relative amount of proteins in each band was quantified using ImageJ (NIH), and normalized to β-actin (TA-09, ZSGB-BIO) as an internal loading control.