The Very Atypical ISRIB Story

35 The averaged expression of the PDGF�� receptor (PDGF��R) ligands PDGF-B and PDGF-D was calculated using the arithmetic mean of PDGF-B and PDGF-D expression levels. A total of 1.2 �� 106 BJhTERT fibroblasts were cultured in 10-cm dishes in Dulbecco��s modified Eagle��s medium (Gibco Life Technologies, Gergy-Pontoise, France) supplemented with 1% heat-inactivated fetal calf serum, 2?mmol/L l-glutamine, 100 U/mL penicillin, and 100 ng/mL streptomycin and kept Inhibitor Library manufacturer at 37��C in a 5% CO2 humidified atmosphere for 24 hours. Cultures were incubated with or without 20 ng/mL PDGF-B (Peprotech, Rocky Hill, NJ) for another 24 hours before harvest. Total RNA from three replicates of each PDGF-B�Cstimulated and non-stimulated Tasisulam control cell was extracted using the TRIzol (Life Technologies Europe BV, Stockholm, Sweden) and the RNeasy Mini Kit (Qiagen Inc., Hilden, Germany), followed by purification using the RNeasy micro kit (Qiagen Inc.) and hybridization to HumanHT-12, version 3.0, Expression BeadChips (Illumina Inc., San Diego, CA) at the Swegene Center for Integrative Biology at Lund University (Lund, Sweden). Data management and normalization were performed using BeadStudio, version 3.1.3.0, Software (Illumina Inc.). Raw intensity values represent mean bead pool intensity values for each probe. Data were normalized using a quantile normalization algorithm, as implemented in BeadStudio, version 3.1.3.0, Software. Probes were mapped to RefSeq Release 22 (National Center for Biotechnology Information Build 36.2) and UniGene Build 199. Only probes with an annotated gene symbol were used in further analyses. Probe identifiers were mapped to Entrez gene identifiers, according to manufacturer��s annotation files. Genes differentially expressed between fibroblasts stimulated with PDGF-B and control cells were identified with the R MWT package version ISRIB manufacturer 0.2.6.36 The MWT package implements a moderated Welch test that accounts for unequal group variances, making it suitable for experiments with a low sample number. In case of multiple probe sets corresponding to the same gene ID, the one with the most significant P value was kept. These P?values were adjusted for multiple testing using the false-discovery rate (FDR) with the procedure outlined by Benjamini and Hochberg. 37 All genes with an FDR