Fig 1: FLRT2 activates Akt/mTOR signaling via the binding of UNC5B with Rac1. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the upregulated differentially expressed genes (DEGs) in PMs of Flrt2fl/fl and Flrt2ΔMyel mice. (B) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), and S6 proteins in PMs isolated from Flrt2fl/fl and Flrt2ΔMyel mice i.p. injected with thioglycollate for 3 days (n = 3 mice per group). (C) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), S6, Flag-FLRT2, and FLRT2 protein levels in THP-1 cells transfected with control or hFLRT2 vector for 48 h (n = 3). (D) Immunoblot analysis of the indicated protein levels in THP-1 cells transfected with shNC or shFLRT2 #3 vector for 48 h (n = 3). (E) Immunoprecipitation samples were collected using anti-Flag antibody from control vector and Flag-UNC5B-overexpressing HEK293T cells and subjected to mass spectrometry (MS) to identify potential binding partners of UNC5B protein. A unique peptide of Rac1 protein was identified in the UNC5B-overexpressing immunoprecipitation samples by analyzing the mass-to-charge ratio of the samples. (F) The endogenous interaction between Rac1 and UNC5B was confirmed in THP-1 cells by co-immunoprecipitation (co-IP) analysis. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 2: UNC5B mediates FLRT2-promoted THP-1 cell maturation into macrophages. FLRT2 was overexpressed by transfecting the hFLRT2 vector, and UNC5B was silenced by transfecting UNC5B-specific shRNA into THP-1 cells. (A) Representative phase-contrast light microscopy images showing morphological alterations of THP-1 cells transfected as indicated (n = 3). Scale bar, 50 μm. (B) qPCR analysis of Itgam, Csf1r, Cd36, Msr1, and Olr1 mRNA in THP-1 cells transfected as indicated (n = 3). (C) Immunoblot analysis of CD36, SR-A, UNC5B, and GFP-FLRT2 proteins in THP-1 cells transfected as indicated (n = 3). (D) HUVECs expressing RFP were co-cultured with THP-1 cells transfected as indicated. After 6 h co-culture, pictures were taken using a fluorescence microscope (n = 3). Scale bar, 5 mm. (E) Transwell cell migration assay was performed in THP-1 cells transfected as indicated, and the numbers of the migrated cells were counted (n = 3). Scale bar, 100 μm. (F) Phagocytosis assays were performed by culturing the cells transfected as indicated in Texas red-conjugated zymosan particles for 2 h at 37°C (n = 3). Cells were observed for internalization of the particles by fluorescence microscopy. Scale bar, 50 μm. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 3: Schematic diagram of our major findings. Pro-differentiation factors, such as PMA, M-CSF, and thioglycollate, upregulate Flrt2 expression in monocytes. FLRT2, in turn, accelerates monocyte-to-macrophage differentiation by binding to UNC5B via its ECD and subsequently activating the Akt/mTOR signaling pathway.
Fig 4: The direct interaction of the FLRT2 ECD with UNC5B is required for FLRT2-promoted THP-1 cell differentiation into macrophages. (A) Myc-tagged hUNC5B and/or Flag-tagged hFLRT2 were transfected into HEK293T cells. Immunoprecipitation samples were collected using anti-Flag antibody, followed by immunoblotting for Myc and Flag (n = 3). (B) Immunolabeling of FLRT2 and UNC5B in PMA-treated THP-1 cells showing the colocalization of endogenous UNC5B and FLRT2. Scale bar, 10 μm. (C) UNC5B was immunoprecipitated from PMA-incubated THP-1 cells, followed by immunoblotting for FLRT2 and UNC5B (n = 3). (D) Schematic illustration of FLRT2 and UNC5B constructs. LRRNT, leucine-rich repeat N-terminal domain. LRR, leucine-rich repeat. LRRCT, leucine-rich repeat C-terminal domain. FNIII, fibronectin type III domain. (E) UNC5B interacts with hFLRT2-ECD but not hFLRT2-ICD (n = 3). (F, G) THP-1 cells were transfected with control, human FLRT2 extracellular domain (hFLRT2-ECD) or human FLRT2 intracellular domain (hFLRT2-ICD) vector for 48 h, followed by qPCR (F) and immunoblot (G) analyses. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t-tests. NS, not significant. ** P < 0.01, **** P < 0.0001.
Supplier Page from Sino Biological, Inc. for Human UNC5B / UNC5H2 Gene ORF cDNA clone in cloning vector