ABSTRACT: Gene expression analysis of untreated and NT-shRNA transduced kidney proximal tubular epithelial cells (PTECs) at day 6 and day 16 after transduction, as well as SETD2-shRNA transduced PTECs at day 25 after transduction
Project description:Primary kidney PTECs gradually became senescesince day 16, but SETD2 depletion prevented PTECs from senescence and maintained their proliferation beyond their limited dividing capacity. Transcriptional profiling of human PTECs, with comparing of non-senescent PTECs (PTECs-day 6), senescent PTECs (PTECs-day16), and SETD2 depleted PTECs at day 25 (SETD2 KD-PTECs-day 25). Three PTECs of different origins were transduced with shRNA constructs against SETD2 (sh1 or sh2), or with a non-targeting sequence. Untreated and NT-shRNA transduced samples were harvest at day 6 and day 16 respectively, SETD2-KD shRNA transduced PTECs were harvest at day 25.
Project description:We used cDNA microarray analysis to identify the Stat5a/b transcriptome in AR-depleted CWR22Pc cells. We sequentially depleted AR protein (AR knockdown) followed by Stat5a/b (Stat5a/b knockdown) using lentiviral shRNA in CWR22Pc cells. CWR22Pc cells were transduced with scramble shRNA (shCtrl), shAR or shStat5a/b at the start of the experiment (day 0), followed by transduction with a second lentiviral shRNA, either shCtrl or shStat5a/b, 3 days later (day 3). CWR22Pc cells were harvested on day 6 for analysis of gene expression profiles.
Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct.. In this part of the study pooled shRNAs were transduced into MDA-MB-231 breast carcinoma cell lines and their inhibitory effects four weeks post transduction were analyzed via microarray hybridization.
Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct.. In this part of the study pooled shRNAs were transduced into MDA-MB-231 breast carcinoma cell lines and their inhibitory effects four weeks post transduction were analyzed via microarray hybridization.
Project description:To determine the effect of darunavir upon HIV-induced gene regulation in renal epithelial cells, immortalized human renal tubular epithelial cells (HPT1b) were transduced with VSV-pseudotyped gag/pol-deleted HIV lentivirus or EGFP control lentivirus. 4 days later, cells were treated with darunavir (3uM) or vehicle control from 4-7 days post transduction. Primary comparisons in gene expression were between HIV and control-transduced cells at 4 days post transduction, and between DRV and control-treated HIV-transduced cells at 7 days post transduction.
Project description:Transcriptional profiling of primary cutaneous anaplastic large cell lymphoma cell line Mac-1 cells transduced with lenti-virus vector harboring shRNA against SATB1 gene comparing control untreated Mac-1 cells and Mac-1 cells transduced with scrambled shRNA, in which SATB1 expression is not affected.
Project description:C-peptide exerts beneficial effects on glomerular hyperfiltration in type I diabetic patients. As C-peptide localizes to the nucleus, we investigated the transcriptional activities of C-peptide in proximal tubular cells isolated from diabetic rats. Two groups of proximal tubular cells isolated from type I diabetic rats: 1 treated with C-peptide, and 1 untreated. 2-3 replicates per group.
Project description:The metastatic melanoma cell line A375SM was stably transduced with NFAT 1 shRNA utilizing the pSIH-HI- 5 copGFP lentiviral vector. A non-targeting (NT) shRNA construct with no known homology to any human gene was was created and transduced into A375SM cells as a control. These two sets of cells were then subjected to microarray profiling using the Human OneArray microarray (version HOA 6.1, GEO Platform GPL19137) from Phalanx Biotech Group. NFAT shRNA A375SM cells vs. NT-shRNA A375SM cells were subjected to microarray analysis. 3 biological replicates for each cell type were used.
Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).
Project description:RNA sequencing of nucleus pulposus cells transduced with shRNA (control or TonEBP-targeted) and either untreated or treated with TNF-a (24h)
