2); BGN (Bgn, Gene ID: 12111) (forward 5′-TAGGAAAGATGGATAGACCACAC-3′; reverse 5′-GAACTTGTTGAAGAGAGAACACC-3′; amplicon with 145-bp, GenBank NM_007542.4). The reaction Ku 0059436 solution was carried out in 96-well plates with a final volume of 20 μL, containing 1 μL of cDNA, 1 μL of probe or set of primers (5 pmol), 10 μL of Jump Start SYBR Green Taq Ready Mix and 8 μL of Nuclease-Free water. The SYBR Green amplification conditions consisted in a initial denaturation of 5 min at 95 °C, followed by 40 cycles of 15 s at 95 °C (denaturation), 30 s at 54 °C (COL1); 59 °C (MMP-2; BIGL); 60 °C (ALP); 60 °C (DSPP) (annealing temperature),

and 30 s at 72 °C (extension). The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected, denoted Cp (Crossing point). Target genes expression were normalized by the reference housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Gapdh, Gene ID: 14433) (forward 5′-GTGCTGAGTATGTCGTGGAGT-3′; reverse 5′-TTGTCATATTTCTCGTGGTTCA-3′; amplicon 154-pb, GenBank NM_008084.2) and the mean value for the Control group was

set to 100% of mRNA expression and served as a reference. To evaluate whether PTH administration affect the MMP-2 secretion, MDPC-23 cells were cultured as previously describe in 96-well plate (n = 4). At the end experimental period (3 cycles × 48 h), the cells were washed with PBS and cultured in the absence of FBS for 24 h Tangeritin at 37 °C in an atmosphere of high humidity and 5% Bleomycin clinical trial CO2 for MMPs secretion. Then, cell culture medium (DMEM) containing the secreted MMPs was collected and frozen at −70 °C. After thawing the protein concentration was determined by a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA), using the Bradford method. Fifteen nanograms of the each sample was mixed with non-reducing sample buffer (2% SDS; 125 mM Tris–HCl, pH 6.8, 10% glycerol, and 0.001% bromophenol blue) and resolved in 10% sodium dodecyl sulphate-polyacrylamide gels copolymerized with 1.6 mg/mL of gelatin (Sigma–Aldrich, St. Louis, MO, USA) as substrate. Protein

renaturation was done by incubation of the gels in 2% Triton X-100 (Sigma–Aldrich, St. Louis, MO, USA), and then the gels were immersed in activation buffer (50 mM Tris–HCl, pH 7.4, 5 mM CaCl2) for 16 h at 37 °C. Gelatinolytic activity was detected after staining with Coomassie Brilliant Blue R250 (Bio-Rad Laboratories, Hercules, CA, USA). To confirm that the bands were related to MMP-2 activity, a molecular weight was used and also control reaction was made to inhibit the gelatinolytic activity by adding 2 mM of 1.10-phenanthroline (Sigma–Aldrich, St. Louis, MO, USA), a nonselective zinc chelator, to the activation buffer, confirming the specificity of the reactions. The gel image was obtained by Gel Logic 212 PRO (Carestream Health, Inc., USA) using a Molecular Image Software 5.