Project description:This study aimed to understand the role of ILF2 upregulation in metastatic melanoma cutaneous progression and DNA damage response. The goal of RPPA analysis was to determine the protein expression profiles in DP-0574 and IM-0223 melanoma cell lines with ILF2 overexpression (ILF2-OV) or control empty vector (EV). By analysis of RPPA in both metastatic melanoma cell lines, we found that ILF2-OV controls significantly increased RAD50 expression.
Project description:Here, we systematically profile the methylation sensitivity of 18 human TFs spanning 11 structural families using chemically synthesized DNA libraries containing position-specific 5-methylcytosines (5mC) in CpG, non-CpG, and hemi-methylated contexts, measured via high-throughput protein-binding microarrays. Our results reveal extensive TF sensitivity to methylation state, position, and strand orientation—including strong binding of several TFs to non-CpG and hemi-methylated sites. The presence of 5mC can dramatically alter TF-DNA interactions: transforming low-affinity sites into high-affinity ones by enabling new contacts, or silencing otherwise favorable motifs through steric hindrance. Genomic analyses further show that the methylation-sensitive sequences identified in vitro are represented within enhancers and regulatory elements, exhibiting distinct methylation patterns across cell types. Together, our findings uncover a previously hidden layer of methylation-dependent TF-DNA recognition, broadening the understanding of epigenetics in transcriptional regulation.
Project description:Here, we systematically profile the methylation sensitivity of 18 human TFs spanning 11 structural families using chemically synthesized DNA libraries containing position-specific 5-methylcytosines (5mC) in CpG, non-CpG, and hemi-methylated contexts, measured via high-throughput protein-binding microarrays. Our results reveal extensive TF sensitivity to methylation state, position, and strand orientation—including strong binding of several TFs to non-CpG and hemi-methylated sites. The presence of 5mC can dramatically alter TF-DNA interactions: transforming low-affinity sites into high-affinity ones by enabling new contacts, or silencing otherwise favorable motifs through steric hindrance. Genomic analyses further show that the methylation-sensitive sequences identified in vitro are represented within enhancers and regulatory elements, exhibiting distinct methylation patterns across cell types. Together, our findings uncover a previously hidden layer of methylation-dependent TF-DNA recognition, broadening the understanding of epigenetics in transcriptional regulation.
Project description:Aberrant activation of PI3K pathway is frequently observed in triple negative breast cancer (TNBC). However single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor BKM120. Baseline and post-treatment PDX tumors harvested following 3 days of BKM120 or vehicle administration were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive PDX model at the completion of approximately 3-4 weeks of treatment. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increase in the levels of phosphorylated forms of Aurora kinases. Sensitivity to BKM120 was associated with higher baseline levels of proapoptotic markers (Bak and Caspase 3) and a greater number of markers differentially changed following BKM120 therapy. Interestingly, markers indicating PI3K pathway signaling activation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.
Project description:We performed autoantigen arrays on sera derived from Sjogren's syndrome mice that lacked Myd88 systemically (NOD.B10LSL-/- strain) or lacked Myd88 in specific tissues (NOD.B10Myd88delta and NOD.B10LSL-/-Vav+ strains). We also examined sera from Myd88-sufficient controls (NOD.B10Myd88fl/fl, NOD.B10, and NOD.B10LSL+/- strains).
Project description:Plasma protein expression patterns can be used as prognostic biomarkers in various types of cancer. We aimed to identify a protein-based signature for distant metastatic risk assessment in patients with locoregionally advanced nasopharyngeal carcinoma (LA-NPC).
Project description:Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components, and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct co-culture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial Least Squares Regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP, and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52 ± 3.65 fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplify hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route towards identifying feeder-free culture platforms for HSC expansion.
Project description:Mycobacterium tuberculosis (Mtb) antigen-specific cellular response is promising for detectionof Mtb infection, but not efficient for diagnosis of TB. We firstly identified 16 TB disease-specific protein markers measured in the culture supernatant of Mtb-stimulated whole blood using a 640 human proteins array, the highest throughput antibody-based protein array available at the time when we did this study. Potential TB-related proteins were then analyzed across three different patient cohorts comprised of healthy controls, LTBI, non-TB pneumonia, and TB patients to evaluate how the biomarkers performed in diagnosing TB in the real clinical setting. The data finally reveal an eight-protein biosignature of TB.
Project description:β-cell specific IFT88 knock-out mice recapitulate human diabetes with impaired insulin secretion and altered islet hormone paracrine regulation. To examine the signaling pathways regulating islet cell function, we subjected protein lysates of whole islets from control and IFT88 knockout mice to a commercial signaling-protein array analysis (Full Moon Bio, Inc). Samples were probed against 1358 antibodies with 2 replicates per antibody on 76 x 25 x 1mm glass slides.
Project description:To compare the cell signaling events between PC-3 cells and MDA-MB-468 cells, we performed a Reverse Phase of Protein Array (RPPA) profiling on 468 and PC-3 Cells that treated with DMSO, AZD5363, MS21 at 1µM for 24hr respectively.