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Cultured cell LC-MS Analysis to check PTM

ABSTRACT: To figure out relation between KRAS and AIMP2-DX2(an exon 2-deleted splice variant of AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2) based on its stability

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Lung Cancer

SUBMITTER: Dae Gyu Kim

LAB HEAD: Sunghoon, Kim

PROVIDER: PXD029839 | Pride | 2022-03-31

REPOSITORIES: Pride

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Project description:Purpose: The goal of this study is to identify genes and pathways in macrophages that may be altered by hepatocytes with replicating HBV Methods: THP-1 monocytic cells were treated with PMA for differentiation into macrophages (M0). These macrophages were co-cultured for 4 days with HepAD38 cells with (HBV-) or without (HBV+) the treatmetn of tetracycline and then lysed for the isolation of RNA using NEBNext Ultra II RNA Library Prep Kit for Illumina. The polyA+ RNA-seq was conducted in triplicate, and about 30 million sequence reads per sample that passed quality filters were analyzed by Partek Flow. The qRT–PCR validation of the results was performed using SYBR Green assays. M0 macrophages without co-culturing with HepAD38 cells were also analyzed to serve as the control (M0). Results: To understand how HBV might affect THP-1 macrophages and how macrophages suppressed HBV replication, we conducted the RNA-seq analysis to determine the gene expression profiles in THP-1 macrophages (Mϕ) without co-culturing with HepAD38 cells (M0), THP-1 macrophages co-cultured with HepAD38 cells in the presence of tetracycline (HBV-), and THP-1 macrophages co-cultured with HepAD38 cells in the absence of tetracycline (HBV+). The analysis of RNA-seq results revealed 3946 differentially expressed genes (DEGs) between Mϕ(HBV+) and Mϕ(M0) cells, 3333 DEGs between Mϕ(HBV-) and Mϕ(M0) cells, and 783 DEGs between Mϕ(HBV+) and Mϕ(HBV-) cells (Figure 2A). The heatmap analysis revealed that Mϕ(HBV+) cells expressed significant higher levels of M1 markers including IL-1β and lower levels of M2 markers than in Mϕ(HBV-) cells (Figure 2B). The induction of IL-1β in Mϕ(HBV+) cells was also highly prominent when the cytokine expression profiles were analyzed by the volcano plot (Figure 2C). This induction of IL-1β was confirmed by RT-qPCR analysis (Figure S2E). These results indicated that THP-1 macrophages co-cultured with cells containing replicating HBV would undergo the M1 polarization. Interestingly, the heat map analysis of top 20 DEGs, based on low false discovery rate and high Log2 fold changes, between Mϕ(HBV+) and Mϕ(HBV-) cells revealed that seven of them were mitochondria-encoded genes that regulate the OXPHOS (Figure 2D). The pathway analysis indicated that the OXPHOS pathway was indeed upregulated in MΦ(HBV+) cells, although some pathways in these cells were also downregulated (e.g., Sirtuin signaling) and some were not affected (e.g., insulin receptor signaling) (Figure 2E). Conclusions: Our study provided the detailed analysis of the response of macrophages to hepatocytes containing replicating HBV. Our results indicated that HBV-could induce the M1 polarization of THP-1-derived macrophage. The induction of CXCL1 expression in macrophages by HBV likely plays an important role in the hepatic infiltration of neutrophils, whih express a high level of CXCR1 , the receptor of CXCL1. Our finding of an increased expression of mitochondria-encoded genes that regulate OXPHOS also suggested that HBV might promote OXPHOS in macrophages.

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Cultured cell LC-MS Analysis to check PTM

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