Briefly, cell cultures were carried out as previously described, and RNA extraction and reverse-transcription Venetoclax research buy were performed using Trizol Reagent and Ready-To-Go First Strand kit (see section on RNA isolation and gene expression by real-time PCR). The PCR reaction was performed as previously described, in a 50 μL reaction mixture containing 5 μL complementary DNA, 20 mM Tris-HCl, 50 mM MgCl2, 200 μM of each deoxynucleotide triphosphate, 0.3 mM of each specific primer (sense: 5′-TCA CAC TCC TCG CCC TAT T-3′ and antisense: 5′-CGA TGT GGT CAG CCA ACT-3′), and 0.03 U/μL Taq DNA polymerase (GibcoBRL, Grand Island, NY). PCR of β-actin was used as an endogenous control. The expected sizes of the PCR products of osteocalcin

and β-actin were 246 and 285 base pairs, respectively. A pool of primary osteoblastic cells from 10 donors were plated in 24-well tissue

plates and were incubated in DMEM/HAM F-12 (1:1) medium, supplemented with 10% of FBS and 10 μg/mL of ascorbic acid. In order to synchronize after reaching osteoblast subconfluence, culture medium was replaced with DMEM/HAM F-12 containing 100 μg/mL of ascorbic acid and incubated for 24 hours. Cells were subsequently incubated for 24, 48, and 72 hours with different concentrations of unconjugated bilirubin (10, 50, 100, and 1000 μM) or pooled samples from cholestatic patients with normal and high bilirubin levels, and pooled samples from healthy controls, in DMEM/HAM F-12 medium with 10 μg/mL ascorbic acid. Cell viability was measured in duplicate using a colorimetric assay based on the cleavage of the tetrazolium salt U0126 purchase WST-1 by mitochondrial dehydrogenase in viable cells (Cell Proliferation Reagent WST-1; Roche, Basel, Switzerland). The absorbance was read at 450 nm wavelength with an enzyme-linked immunosorbent assay reader. Osteoblast 上海皓元医药股份有限公司 differentiation was measured by the determination of alkaline phosphatase activity. Briefly, primary osteoblasts from three subjects were plated in 12-well tissue plates and incubated in supplemented

medium. After synchronization, cells were incubated for 24, 48, and 72 hours in 10 μg/mL of ascorbic acid with different concentrations of unconjugated bilirubin (10, 50, 100, and 1000 μM) or pooled samples from patients with normal and high bilirubin levels, and samples from healthy controls. Then, cells were washed with phosphate-buffered saline and lysed with a lysis buffer (CelLytic M; Sigma Aldrich). Cell extracts were incubated with 2 mg/mL of p-nitrophenylphosphate (pNPP) in a 0.05 M glycine buffer containing 0.5 mM MgCl2 (pH 10.5) at 37°C for 30 minutes. The reaction was stopped by the addition of 0.4 N NaOH to the reaction mixture, and the alkaline phosphatase activity was quantified by absorbance at 405 nm. Total protein content was determined with Bradford’s method in aliquots of the same samples with the Quick Start Bradford Protein Assay (Bio-Rad Laboratories, Madrid, Spain).