Project description:To explore the potential functions of ASAP2 in macrophage, we established ASAP2 stable knockdown THP-1 cells and differentiated them into macrophages with PMA (50 ng/ml, 48 hours). Then we performed RNA-sequencing on the macrophages derived from THP-1 with (shASAP2) or without ASAP2 knockdown (shNC) to analyze the enriched functions by ASAP2-correlated genes and differentially expressed genes.
Project description:Purpose: The aim of this study was to obtain a comprehensive transcriptomic analysis of IL-26-exposed macrophages. Methods: THP-1 derived macrophage were generated by incubating THP-1 cells with PMA (50 ng/ml) for 24h.Then THP-1 macrophages were treated with IL-26 for 24h, which were collected and applied to RNA-sequencing. The constructed sequencing library was sequenced using an Illumina Novaseq 6000 system. The expression of transcripts was calculated as fragments per kilobase of exon model per million mapped reads (FPKM). Results: In total, we identified 1303 differentially expressed protein-coding genes between untreated and IL-26-treated macrophages, including 667 up-regulated and 636 down-regulated genes.
Project description:Human monocyte THP-1 cells obtained from ATCC were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% FBS and supplemented with 10 mM Hepes (Gibco BRL). THP-1 was differentiated into macrophages by 24-h incubation with 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) followed by 24-h incubation in RPMI medium. Macrophages were further polarized to M1 macrophages by incubation with 10 pg/ml of lipopolysaccharide (LPS; Sigma) and 20 ng/ml of interferon (IFN)-γ (R&D Systems, MN) and are referred to as M(LPS+IFN-γ) cells. M2 macrophages were obtained by incubation with 20 ng/ml of interleukin (IL)-4 (R&D Systems) and are referred to as M(IL4) cells. To test the represented polarization marker of PMA differentiated-THP-1 macrophages stimulated with 20 ng ml(-1) IFNγ + 10 pg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vetro into the M1 and M2 state, respectively. We used microarrays to detail the gene expression pools to identify distinct M1 and M2 state during this process.
Project description:MSCV-GFP-IRES (IRES), MSCV-GFP-Myc-NIC1 (NIC1) and MSCV-GFP-DNMAML (DNMAML) were retrovirally transduced in to THP-1 cell (pCL-Ampho was used as packaging plasmid). GFP positive cells were sorted for selective desired cell. Then IRES, NIC1 and DNMAML overexpressing THP-1 were pretreated with PMA (5ng/ml) for 48 h before stimulation with IL-4 (20 ng/ml) for 3 h.
Project description:We present a genome-wide map of RNA Polymerase III subunit localization in human THP-1 monocytes and THP-1 derived macrophages after 72 hr exposure to PMA, as well as profiles of POLR3G and POLR3GL occupancy in THP-1 monocytes after 4 hr exposure to Pol III drug inhibitor ML-60218; 27 uM
Project description:We showed that co-culture with TAMs triggered Bmi1 expression in gastrointestinal cancer cell lines. miRNAs have been found to target various oncogenes and tumor suppressors. We therefore hypothesized that the regulation of Bmi1 expression in gastrointestinal cancer cells may be mediated by miRNAs using miRNA microarray analysis. THP-1 cells were seeded in the transwell inserts (3540, Corning) for 6-well plates (1 M-CM-^W 106 cells/well). For preparation of M1-polarized THP-1 macrophages, 320 nM phorbol myristate acetate (PMA) was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interferon (IFN)-M-NM-3 and 100 ng/ml lipopolysaccharide for the following 18 h. For preparation of M2-polarized THP-1 macrophages, 320 nM PMA was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interleukin (IL)-4/IL-13 for the following 18 h. After three washes to remove cytokines, M1- or M2-polarized THP-1 macrophages were co-cultured in upper inserts with AGS cells in 6-well plates (1 M-CM-^W 105 cells/well) without direct contact, in each medium without 10% FBS as described above. After 24 h of co-culture, the upper inserts containing macrophages were discarded. AGS cells were collected and analyzed to identify downregulated microRNA in a gastric cancer cell line co-cultured with M1- or M2-polarized macrophage.
Project description:To analyse gene expression differences between differentiated bronchial epithelial cells kept under control conditions or treated for 72 hours with cytokines (TNFalpha10 ng/ml; IL17 20ng/ml)
Project description:MiRNAs play pivotal roles in regulating macrophage functions, including differentiation, polarization, recruitment, and activation of inflammation. in this review, to provide novel insight into macrophage differentiation-specific mitomiR signatures and associated mitochondrial pathways, we performed a global miRNA profiling on extracted mitochondria and total cell content of human macrophages. The THP-1 monocytic cell line was treated with phorbol-12-myristate-13-acetate (PMA) to induce differentiation into macrophages The human monocytic cell line THP-1 cells were differentiated into THP-1 derived macrophages with 40 ng/mL phorbol 12-myristate 13-acetate (PMA),The mitochondrial fraction of the cells were enriched and purified using anti-TOM22 microbeads (Miltenyi Biotec). After total RNA extraction, and using Taqman low-density arrays (TLDA), we performed the global miRNA profiling on extracted mitochondria and compared with total cell content
Project description:To analyse gene expression differences between differentiated bronchial epithelial cells kept under control conditions or treated for 72 hours with TNFalpha+IL-17 (10 ng/ml and 20ng/ml respectively)