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:Primary kidney PTECs gradually became senescent after a few passages, but SETD2 depletion prevented PTECs from senescence and maintained their proliferation beyond their limited dividing capacity. Transcriptional profiling of human PTECs, with comparing of SETD2 wild-type non-senescent PTECs at day 6 (WT-PTECs-day 6), SETD2 wild-type senescent PTECs at day 16 (WT-PTECs-day16), and SETD2 knockdown PTECs at day 25 (KD-PTECs-day 25).
Project description:We performed bulk RNA sequencing to characterize proximal tubular epithelial cells (PTECs) and PTECs that are positive for CD24 and CD133 (presumed to be scattered tubular cells (STCs)). We found an upregulation of many STC markers in our bulk RNA sequencing data and identified a pro-inflammatory and pro-fibrotic phenotype for STCs compared to PTECs. Many forms of metabolism were downregulated in the STCs compared to PTECs.
Project description:Transcriptional profiling of human WT-PTECs, SETD2-KD PTECs, PBRM1-KD PTECs, and 10 different ccRCC derived cell lines. ccRCC derived cell lines showed distict expression signatures as compared to PTECs, some of them also present in SETD2-KD PTECs, and/or PBRM1-KD PTECs.
Project description:To identify RNA transcripts involved in acute and chronic renal epithelial injury, we performed unbiased whole transcriptome profiling of human proximal tubular epithelial cells (PTECs) in hypoxic and inflammatory conditions. RNA sequencing (RNA-seq) revealed that the protein-coding and noncoding transcriptomic landscape differed between hypoxia-stimulated and cytokine-stimulated human PTECs.
Project description:Retinoic acid (RA) signaling is activted in proximal tubular epithelial cells (PTECs) after acute kidney injury (AKI). In these studies we evaluated the functional role of this by inducing ischemia reperfusion-induced AKI (IRI-AKI) in mice after inhibiting RA signaling in PTECs genetically. Here we evaluated the effects of this on the activation of renal mononuclear cells (MNCs) by evaluating RNA expression in CD11B+ cells isolated from kidneys after IRI-AKI. Compared with control mice, there was a reducion in expression of inflammatory marker gene expression 3 days after IRI-AKI in mice with inhibition of RAR signaling in PTECs. These findings suggest a mechanism by which inhibition of RAR signaling in PTECs is protective against AKI.
Project description:Ischemia/reperfusion-induced acute kidney injury (AKI) occurs in several clinical conditions accompanied by inflammation, but the underlying mechanisms remain elusive. The Wnt inhibitor Tiki2 is highly expressed in the kidney with unknown function. Here, we demonstrated that Tiki2 is highly expressed in proximal tubular epithelial cells (PTECs) and is dynamically regulated in AKI. Tubular epithelial cell-specific ablation of Tiki2 aggravated immune cell infiltration and tubular injury in IR-induced AKI. Mechanistically, Tiki2 ablation increased the activity of Wnt5a/Ror signaling, which is activated in PTECs in IR-induced AKI, leading to increased active JNK/ERK signaling and the secretion of chemokines and cytokines to promote the infiltration of immune cells. Furthermore, the increased severity of injury and inflammation caused by Tiki2 ablation could be relieved by knocking down Ror2 in PTECs. Our study reveals the critical roles of Wnt5a/Ror signaling and its negative regulator Tiki2 in IR-induced AKI, which are potential therapeutic targets for treating AKI
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.
