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Quantitative label-free proteomic analysis of Wdr47+/+ and Wdr47-/- mouse motile cilia

ABSTRACT: Two 75-cm2 flasks of ependymal cells derived from ten E18.5 Wdr47+/+ or Wdr47-/- embryos at the day 10 post serum starvation were harvested and motile cilia were purified for quantitative label-free proteomic analysis.

ORGANISM(S): Mus Musculus

SUBMITTER: Xueliang Zhu

PROVIDER: PXD028219 | iProX | Tue Sep 07 00:00:00 BST 2021

REPOSITORIES: iProX

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Publications

GLUT3 as an Intersection of Glycerophospholipid Metabolism and the Innate Immune Response to <i>Candida albicans</i>.

Wu Xian X Zhang Ge G Yang Wen-Hang WH Cui Jing-Tao JT Zhang Li L Xiao Meng M Xu Ying-Chun YC

Frontiers in cellular and infection microbiology 20210618

Immune cells can optimize the management of metabolic resources to balance their energy requirements in order to regulate immune responses. The interconnection between immunometabolism and fungal infections is becoming increasingly apparent. Using proteome and metabolome assays, we found that stimulation of primary human monocytes by <i>Candida albicans</i> was accompanied by upregulation of glucose transporter 3 (GLUT3) and activation of the glycerophospholipid metabolism pathway. Upregulated G ...[more]

PMID: 34222036

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Proteomics of mouse ependymal cilia

Project description:Two 75-cm2 flasks of ependymal cells derived from ten E18.5 C57BL/6J mouse embryos at the day 10 post serum starvation were harvested and motile cilia were collected for mass spectrometry analysis.

2021-10-18 | PXD029174 |

Chikungunya virus phosphoproteomics

Project description:SILAC-labeled MRC-5 cells were seeded in 75-cm2 flasks 1 day before infection with CHIKV-LS3 [1] at MOI 5. One hour post infection (h p.i.), the inoculum was removed and replaced with SILAC DMEM containing 2% dialyzed FBS, 0.280 mM arginine, 0.384 mM lysine, 0.5 mM proline, 25 mM HEPES, 2 mM L-Glutamine and 1% NEAA. At 2, 8, and 12 h p.i., infected and mock-infected cells were harvested for phosphoproteomics analysis by lysis in 4% SDS, 0.1M Tris pH 7.6, followed by heating to 96°C for 10 min. At 12 h p.i,. protein lysates for western blot (WB) analysis were harvested in 4× Laemmli sample buffer (LSB) (100 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 40 mM DTT, 0,04 mg/ml bromophenol blue) and cells grown on coverslips were fixed in 3% PFA in PBS . The experiment was performed in duplicate with a label swap.

2022-02-28 | PXD009381 | Pride

Testing effects of cell density on NGN2 iN transcriptom

Project description:We previously established a standard protocol for NGN2 iN produciton using iP11N iPSC line. Here we aim to explore whether plating density contributes to batch-to-batch variabilities in the transcriptome. Cells were plated at high (400K/cm2) or normal (150K/cm2 densities) and cultured according to the established protocol. Day 28 neurons were harvested and total RNA extracted for bulk RNA sequencing

2024-09-13 | GSE273553 | GEO

The effects of primary cilia on endothelial gene expression exposed to flow

Project description:Biomechanical forces such as wall shear stress induced by flowing blood influence the behavior of vascular cells and have been shown to be crucial in the onset of vascular disease. Primary cilia are increasingly recognized as important biological sensors of low WSS. The goal of the study was to investigate gene expression profile of endothelial cell under low flow (2 dynes/cm2) as compared to physiological flow (30 dynes/cm2) in presence or absence of primary cilia.

2022-05-25 | GSE139580 | GEO

Transcription profiling of Xenopus epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1. FoxJ1 misexpression leads to the induction fo ectopic cilia in Xenopus laevis epithelia. Results show which genes are affected by FoxJ1 during the induction of ectopic cilia. Ciliated cells that produce a leftward fluid flow have been proposed to mediate left-right patterning in many vertebrate embryos. These cilia combine features of primary sensory and motile cilia, but how such cilia are specified is unknown. We address this issue by analyzing the Xenopus and Zebrafish homologs of FoxJ1, a forkhead transcription factor necessary for ciliogenesis in multi-ciliate cells of the mouse. We show that the cilia that underlie left-right patterning on the Xenopus gastrocoel roof plate (GRP) and Zebrafish Kupfferâs vesicle (KV) are severely shortened or fail to form in FoxJ1 morphants. We also show that misexpressing XFoxJ1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that XFoxJ1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that FoxJ1 is a critical determinant in specifying cilia used in left-right patterning. Experiment Overall Design: 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD alone or along with FoxJ1. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.

2008-10-25 | E-GEOD-12613 | biostudies-arrayexpress

miR-129-3p control cilia assembly by regulating CP110 and actin dynamics [miR-129-3p overexpression]

Project description:Primary cilium serves as a cellular M-bM-^@M-^\antennaM-bM-^@M-^] to sense environmental signals. Ciliogenesis requires the removal of CP110 to convert the mother centriole into the basal body. Actin dynamics is also critical for cilia formation. How these distinct processes are properly regulated remains unknown. Here we show that miR-129-3p, a microRNA conserved in the vertebrates, controlled cilia assembly by down-regulating both CP110 and four proteins critical for actin dynamics, Arp2, Toca1, abLIM1, and abLIM3. Consistently, blocking miR-129-3p repressed cilia formation in cultured mammalian cells, whereas its overexpression potently induced ciliogenesis in proliferating cells and extraordinary cilia elongation. Moreover, inhibition of miR-129-3p in zebrafish embryos suppressed cilia assembly in the KupfferM-bM-^@M-^Ys vesicle and pronephric duct, leading to developmental abnormalities including curved body, pericardial oedema, and randomised left-right patterning. Our results thus unravel a novel mechanism that orchestrates both the centriole-to-basal body transition and subsequent cilia assembly via microRNA-mediated posttranscriptional regulations. We want to find the targets of miR-129-3p by overexpressing miR-129-3p oligo or control oligo in hTERT-RPE1 cells. Through microarray analysis we could check the downregulated genes and these genes might be the targets of miR-129-3p. RPE1 cells were transfected with control (Ctrl) or miR-129-3p (M129) oligo for 72h, and harvested for RNA extraction and hybridization on Affymetrix microarrays. Two samples: RPE1-Ctrl, RPE1-M129

2012-05-31 | E-GEOD-34846 | biostudies-arrayexpress

Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

Project description:We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A, Rab3B, Rab3C, Rab3D, Rab8A, Rab8B Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulators RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary cilia formation and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

2017-11-14 | PXD007214 | Pride

Effect of FoxJ1 on expression of cilia genes

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate cells, either alone or along with FoxJ1. FoxJ1 misexpression leads to the induction fo ectopic cilia in Xenopus laevis epithelia. Results show which genes are affected by FoxJ1 during the induction of ectopic cilia. Ciliated cells that produce a leftward fluid flow have been proposed to mediate left-right patterning in many vertebrate embryos. These cilia combine features of primary sensory and motile cilia, but how such cilia are specified is unknown. We address this issue by analyzing the Xenopus and Zebrafish homologs of FoxJ1, a forkhead transcription factor necessary for ciliogenesis in multi-ciliate cells of the mouse. We show that the cilia that underlie left-right patterning on the Xenopus gastrocoel roof plate (GRP) and Zebrafish Kupffer’s vesicle (KV) are severely shortened or fail to form in FoxJ1 morphants. We also show that misexpressing XFoxJ1 is sufficient to induce ectopic GRP-like cilia formation in frog embryos. Microarray analysis indicates that XFoxJ1 induces the formation of cilia by upregulating the expression of motile cilia genes. These results indicate that FoxJ1 is a critical determinant in specifying cilia used in left-right patterning. Keywords: Cilia Induction

2008-09-15 | GSE12613 | GEO

RNA-seq of primary mouse B cell progenitor populations after activation with LPS

Project description:Naive resting B-cells were purified from bone marrow from C57BL/6 mice using flow cytometry sorting. Naive B cells were activated with LPS and harvested 3 hours, 10 hours or 33 hours after the LPS treatment. Expanded activated B cells (CD22+ CD138-) and plasmablasts (CD22- CD138+) were also harvested on day 4 post-stimulation.

2020-12-08 | GSE147496 | GEO

Brain heterotopia formation by ciliopathic breakdown of neuroepithelial and blood-cerebrospinal fluid barriers

Project description:In this study, we found that ablation of genes encoding ciliary transport proteins such as intraflagellar transport homolog 88 (Ift88) and kinesin family member 3a (Kif3a) in cortical radial progenitors led to periventricular heterotopia during late mouse embryogenesis. Conditional mutation of primary cilia unexpectedly caused breakdown of both the neuroepithelial lining and the blood-choroid plexus barrier. Choroidal leakage was partially caused by enlargement of the choroid plexus in the cilia mutants. We found that the choroid plexus expressed platelet-derived growth factor A (Pdgf-A) and that Pdgf-A expression was ectopically increased in cilia-mutant embryos. Cortices obtained from embryos in utero electroporated with Pdgfa mimicked periventricular heterotopic nodules of the cilia mutant.

2023-01-10 | PXD038557 | Pride

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