Currently, there is limited knowledge regarding the role of selleck products 1,25D3 and of the proinflammatory cytokines TNFα and IL-6 on CaSR expression in the colon. Therefore, in the present study, we studied the impact of 1,25D3, TNFα, and IL-6 on transcriptional and translational

regulation of CaSR in two colon cancer cell lines with different proliferation and differentiation properties, mimicking different tumor stages. Caco2/AQ cells are a subclone of the Caco-2 cell line [13]. These carry a truncated APC and a missense mutation of β-catenin, and are able to differentiate spontaneously in culture. In the current study we used highly differentiated, 2 weeks post-confluent Caco2/AQ cells. Coga1A is a cell line derived from a moderately differentiated (G2) colon tumor [14]. These cells this website are heterozygous for truncated APC, without any known β-catenin mutations [15]. Confluent Caco2/AQ and Coga1A cells were treated for 6, 12, 24, and 48 h either with 10 nM 1,25D3, 50 ng/mL TNFα (Sigma Aldrich, USA), 100 ng/mL IL-6 (Immunotools, Germany), or the combination of these compounds. Vehicle treated cells were used as controls. RNA isolation and reverse transcription were performed as described previously [16]. Real time qRT-PCR analyses were performed in StepOne Plus system using POWER SYBR GREEN Mastermix following the manufacturer’s recommendations (Life

Technologies, USA). Data were normalized to the expression of the reference genes: β2M or RPLP0 [17] and [18], and set relative to the calibrator (Clontech, USA) to calculate the ΔΔCT value. Primer sequences for CaSR were: 5′-AGCCCAGATGCAAGCAGAAGG-3′ forward, 5′-TCTGGTGCGTAGAATTCCTGTGG-3′ reverse. Cells were grown on sterile glass cover slips. After treatments cells were fixed with 3.7%

paraformaldehyde in PBS, permeabilized with 0.2% Triton-X (Sigma Aldrich, USA) for 20 min, and blocked with 5% goat serum (Jackson ImmunoResearch, USA). Cells were incubated either with rabbit polyclonal anti-CaSR antibody (1:100, Anaspec, USA) or mouse monoclonal anti-CaSR antibody (1:200, Abcam, UK) for 1 h at room temperature. As negative control we used rabbit or mouse IgG, respectively (Abcam, UK and Life Technologies, and USA). As secondary antibody we used Dylight labeled 549 goat-anti-rabbit or Alexa Fluor 647 goat-anti-mouse IgG (1:500, Vector Laboratories and Life Technologies, USA). Nuclei were stained with DAPI (Roche, Switzerland). Images were acquired using TissueFAXS 2.04 (TissueGnostics, Austria). All statistical analyses were performed with SPSS version 18 and graphs were drawn with GraphPad Prism version 5. In case of non-normal distribution, data were log transformed to achieve normal distribution and then subjected to one way ANOVA, followed by Tukey’s multiple comparisons posttest. p-values smaller than 0.05 were regarded as statistically significant.