Filters were applied based on absent calls in

Filters were applied based on absent calls in KPT-330 chemical structure all replicates for both conditions (untreated versus MSU treated) and for detecting very low maximum signals (≤95th percentile of the global

Absent calls distribution). The Limma method [44] was used to define a set of genes differentially expressed between conditions, and a Benjamini–Hochberg multiple test correction of the false discovery rate was applied [45] (adjusted p-value ≤0.05). Functional analysis was performed on nonredundant probe sets using GeneGo MetaCore™ software to select the most significantly enriched pathways and biological processes (FDR ≤ 0.05). The comparison of gene expression patterns between conditions was conducted using hierarchical clustering with MultiExperiment Viewer software [46, 47], setting Euclidean distance as the dissimilarity measure and average linkage as the linkage method. For each selected pathway or biological process, the heat-maps show the Log2 (Ratio) average expression signal for each gene in the MSU-treated Stem Cells inhibitor condition (WT and Nlrp3−/−) versus their respective untreated controls. The microarray data from this publication have been submitted to the ArrayExpress database ( and assigned the identifier E-MEXP-3858. DNA damage was quantified by single-cell gel electrophoresis (also known as the comet assay, R&D) according to the manufacturer’s instructions. DNA fragmentation was visualized by epifluorescence microscopy

using a FITC filter. At least 100 comets were analyzed on duplicate slides. Data were analyzed using Comet ScoreTM (TriTek Corporation). DNA damage was Akt inhibitor quantified by three observers in a blinded fashion based on the distribution of DNA between the head and the tail according to the following formula: Tail% DNA = 100 − (Head% DNA). Damage was also assessed using the Olive Tail Moment: (Tail mean − Head mean) × (Tail% DNA)/100. Total cellular extracts were prepared by lysing cells in ice-cold RIPA buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 355 mM EDTA, protease inhibitor cocktail (Complete Mini protease inhibitor cocktail, Roche), phosphatase

inhibitor cocktail (PhosStop, Roche), 1 mM β-mercaptoethanol). Equivalent protein extracts (40–60 μg) were denatured by boiling in SDS and β-mercaptoethanol before being separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories). The blots were then blocked and probed with the following antibodies: phospho-histone H2AX (Ser139) (#2577, 1:1000), phospho-ATR (Ser428) (#2853, 1:1500), phospho-p53 (Ser15) (#9284, 1:800), phospho-p53 (Ser20) (#9287, 1:800), and total p53 (1C12, #2524, 1:800) from Cell Signaling; phospho-ATM (Ser1981) (10H11.E12, 05-740, 1:1500) and GAPDH (MAB374, 1:20 000) from Millipore; and a-tubulin (sc-5286, 1:1000) from Santa Cruz. The protein complexes were detected using Western Lightning Enhanced Chemiluminescent Substrate (PerkinElmer Inc.

Comments are closed.