Supplementary Materialstable_1. showed that CSIG modulated the mRNA half-life of Cdc14B, CASP7, and CREBL2. This study shows that expression profiling can be used to identify genes that are transcriptionally or post-transcriptionally altered following CSIG knockdown and to reveal the molecular mechanism of cell proliferation and senescence regulated by CSIG. at 4C. The supernatant was collected, and the protein concentration was decided using the BCA Protein Assay Reagent (Pierce). Total protein (20 ~ 40 g) was subjected to 10 ~ 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was transferred to nitrocellulose membranes (Millipore). After blocking in 5% non-fat dry milk in TBST (10 mm Tris-Cl, pH 7.5, 150 mm NaCl, 0.05% Tween 20), the membranes were incubated with primary antibodies overnight at 4C. The membranes were then washed three times with TBST and then incubated with HRP-conjugated secondary antibodies (Zhongshan Biotechnologies Inc., China) for 1 h at room temperature. Proteins were visualized using chemiluminescent substrate (Millipore) according to the manufacturers instructions. Blots were probed with the following antibodies: anti-CSIG [used as previously explained (7)], anti-p16 (sc-759, Santa Cruz), anti-ESCO1 (ab128312, Abcam), anti-Cdc14B (sc-374572, Santa Cruz), anti-KPNA5 (ab81450, Fargesin Abcam), anti-MAP3K3 (ab40750, Abcam), anti-Cdc2 (E53, Epitomics), and anti-PCNA (BS1289, Bioworld). RNA extraction Total RNA was isolated from HEK293 cells and 2BS cells using an RNeasy Mini kit (Qiagen) according to the manufacturers instructions. The quality of the RNA samples was examined by quantifying the A260:A280 ratio (the minimal acceptable ratio is usually 1.7) and the 28S/18S by visualizing rRNA bands in agarose gel (the minimal acceptable ratio is 1.5). Affymetrix cDNA microarray The microarray screen was performed in triplicate using Affymetrix microarray Human Genome U133 Plus 2.0 chips containing 38,500 genes. Briefly, 15C20 g of biotin-labeled cRNA was fragmented by incubating in a buffer made up of 200 mmol/l Tris acetate (pH8.1), 500 mmol/l KOAc, and 150 mmol/l MgOAc at 95C for 35 min. The fragmented cDNA was hybridized with a pre-equilibrated Affymetrix chip at 45C for 14C16 h. The hybridizations were washed in a fluidic station with non-stringent buffer (6 SSPE, 0.01% Tween 20, and 0.005% antifoam) for 10 cycles and stringent buffer (100 mmol/l 2N-morpholino-ethanesulfonic acid, 0.1M NaCl, and 0.01% Tween 20) for 4 cycles and stained with strepto-avidin phycoerythrin. This was followed by incubation with biotinylated mouse antiavidin antibody and restained with strepto-avidin phycoerythrin. The chips were scanned in an Agilent ChipScanner (Affymetrix Fargesin Inc., Santa Clara, CA, USA) to detect hybridization signals. Baseline analyses were done with AGCC to identify statistically significant gene expression alterations between samples derived from HEK293 cells transfected with siCSIG and siNC, respectively. Because samples were analyzed in triplicates, these results were additionally screened for consistent P by the Students 0.05) to eliminate random sampling errors. Quantitative real-time PCR Real-time PCR analysis was performed in triplicate using the SYBR Green PCR Grasp Mix (Applied Biosystems) on an ABI Prism 7300 sequence detector (Applied Biosystems). Rabbit Polyclonal to MARK3 Each PCR was put together using 96-well MicroAmp Optical plates (Applied Biosystems) with a total volume of 15 l made up of 1.5 l Fargesin cDNA templates, 1 M of each primer, and 7.5 l of 2 SYBR Green Grasp Mix and brought to final volume with RNase-free water. Thermal reaction cycles of 50C for 2 min, 95C for 10 min, and 40 repetitions of 95C for 15 s and 60C for 1 min were used. The data were analyzed using the CT method, normalizing the 0.05 and FC 1.5. Of these 590 genes, 311 (53%) were down-regulated and 279 (47%) were up-regulated (Physique ?(Figure2).2). The majority of the selected genes showed moderate (yet significant) alterations in expression of between 1.5- and 2.0-fold (Table ?2;2; for all those genes, see Table S1 in Supplementary Material). Using adjusted 0.05 and FC 2 as a cutoff, there were totally 121 genes showing differential expression following CSIG knockdown, with 57 genes up-regulated (more than 2-folds increase) and 64 genes down-regulated ( 0.5-folds decrease), respectively (Table ?22). Open Fargesin in a separate window Physique 2 Hierarchical clustering warmth map of the 841 genes with significant differentially expressed.