Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.
Project description:RNA-sequencing analysis was utilized to investigate the impact of VEGFR2 knockdown via siRNA on transcriptome profiling in primary human RPE cells under the glucose depletion condition compared to the control siRNA.
Project description:We have performed different High-throughput sequencing techniques to characterize the chromatin and 3D genome of RPE-1 cells. Some of the samples are performed with RPE cells that are resistant to taxol.
Project description:How retinal pigmented epithelial (RPE) cells degenerate from oxidative stress in age-related macular degeneration (AMD) is incompletely understood. The study's intent was to identify key cytoprotective pathways activated by oxidative stress, and to determine the extent of their protection. Immunohistochemistry was used to identify the unfolded protein response (UPR) and mitochondria in the RPE of AMD samples. Maculas with early AMD had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling in mildly degenerated RPE. RPE cells treated with cigarette smoke extract (CSE), by microarray analysis, had over-represented genes involved in the antioxidant and unfolded protein response, and mitochondrial location. CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, which activated CHOP. CHOP knockdown compromised cell viability after CSE exposure. At the same CSE doses, mitochondria generated superoxide anion and produced less ATP. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP, which elicited RPE epithelial-mesenchymal transition, as suggested by altered ZO1 immunolabeling of RPE flatmounts. Our experiments indicate that RPE cells exposed to oxidative stress respond with a cytoprotective antioxidant and unfolded protein response, but develop mitochondrial impairment that contributed to epithelial mesenchymal transition. With similar responses in the RPE of early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress while the ER elicits a protective response during early AMD. A total of 9 samples were analyzed: 3 control samples, 3 samples treated with 100ug/ml of Cigarette Smoke Condensate, and 3 samples treated with 250ug/ml of Cigarettes Smoke Condensate.
Project description:The plasticity of the retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy (PVR), a defective repair process in humans. In contrast, in the embryonic chick, the RPE can be efficiently reprogrammed to regenerate a complete neural retina after surgical removal and when supplied an exogenous source of FGF2. Here, we analyzed discrete RPE cell populations during early times of transiently reprogrammed (RPE 6 hours post-retinectomy) and reprogrammed (RPE 6 hours post-retinectomy and FGF2 treatment) cells, using laser capture microdissection followed by RNA sequencing (LCM-seq) and computational analysis.
Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.
Project description:To assess the geome-wide similarities between primary fetal retinal pigmented epithelium (RPE) and stem-cell derived RPE, we performed whole genome microarray expression on primary RPE and both embryonic stem cell (ESC) derived RPE and induced pluripotent stem cell (iPSC) derived RPE. We found ES-derived RPE better resembles fetal RPE than iPS-derived RPE. Gene expression was measured in primary fetal RPE, ES-derived RPE, iPS-derived RPE. ES cells and BJ fibroblasts were used as controls.
Project description:We performed next-generation sequencing of cytoplasmic fractions of primary human RPE cells. Cytoplasmic sample were size-fractionated on a Blue Pippin device (Sage Science) for size restriction to 200–800-nt long species to eliminate genomic DNA contamination and embedded Alu elements. We found that Alu S predominated (61% of Alu reads), with lower levels of Alu J (31%) and Alu Y (8%). No significant difference in the overall distribution of these Alu sequences that mapped to RPE-specific 47 and non-RPE-specific genes was observed. However, we did identify a cluster of Alu sequences within 2,000 bp of 7 single-nucleotide variant loci statistically associated with AMD48.
Project description:We obtained induced RPE (iRPE) cells from dedifferentiated induced pluripotent stem cells (De-iPSC-RPE). We analyzed their protein profiles by Mass Spectrum