Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A) The relative mRNA levels of IL6, IL10, or CD206 in THP-1 macrophages were analyzed after treatment with CM of HCT116 KRS-positive (shControl or KRS-WT) or KRS-suppressed (shKRS#2) colon cancer spheroids for 24 hours. The data are presented as the mean ± SD. (B) GAS6, IL8, and other cytokine mRNA levels were analyzed in the 3D spheroids with different KRS expression levels for 24 hours. (C and D) THP-1 monocytes, M1 macrophages, or M2 macrophages were treated with GAS6, IL-8, and/or ANG, before measurement of the levels of CD206 (C) and IL10 (D) mRNA. (E and F) THP-1 M1 macrophages or M2 macrophages were treated with CM (E and F) or GAS6, IL-8, and/or ANG (F) for 24 hours before analysis of the protein levels of TNF-α, IL-10, or arginase by ELISA. The data shown represent 3 different observations. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Dunnett tests. See also Supplemental Figures 4 and 5.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A and B) M1 macrophages in 3D collagen I gels were treated with CM from KRS-positive spheroids or with GAS6, IL-8, and/or ANG for 24 hours, before flow cytometry analysis of CD11b (M1 macrophage marker) and CD206 expression on the cell surface (A) or STAT6 phosphorylation, an index for M2 polarization (B). (C and D) THP-1 M1 macrophages were treated with GAS6, IL-8, and/or ANG for 24 hours, before immunoblots (C) or with the CM of KRS-WT spheroids in the absence or presence of antibodies to neutralize GAS6 and MerTK for 24 hours before IL10 and CD206 mRNA analysis (D). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Dunnett tests (D). The data shown represent 3 different observations

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Flow Cytometry, Marker, Expressing, Phospho-proteomics, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A) Plasma membrane and nucleus fractions were prepared from HCT116 spheroids, before immunoblotting. (B) HCT116 spheroids expressing exogenous KRS-WT or KRS point mutants were time-lapse-imaged for 1 day (1d:00h:00min). Scale bars: 40 μm. (C) The GAS6 mRNA levels from the 3D HCT116 spheroids in the absence or presence of a specific MEK/ERK inhibitor (U0126) or with the KRS S207 mutant (right) were measured by real-time PCR. (D) HCT116 spheroids in 3D gels were processed for immunoblots. (E) The CM from HCT116 spheroids were administered to M1 or M2 macrophages before determination of the TNFA, ITGAX (CD11c), IL1A, and CD206 mRNA levels. (F) The 3D HCT116 spheroids were treated with THP-1 CM (Control) or THP-1 M2 macrophages for 24 hours before GAS6 ELISA analysis. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Dunnett tests (C, E, and F). The data shown represent 3 different observations.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Clinical Proteomics, Membrane, Western Blot, Expressing, Mutagenesis, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A) Putative MiTF or c-Jun binding sites in the GAS6 promoter region. (B) HCT116 spheroids embedded in 3D collagen I gels for 24 hours (shControl, shKRS#2, and shKRS#2 treated with M2 macrophage-CM) were processed for ChIP analysis. Antibodies against MiTF or c-Jun were used to examine binding to regions (1, 2, and 3) in the GAS6 promoter. (C–E) HCT116 spheroids in 3D collagen I gels were treated with the CM of control THP-1 or M2 macrophages, before analysis of KRS expression following cellular fractionations (C), mRNA levels of KRS or GAS6 (D), and protein levels of GAS6 and other aminoacyl-tRNA synthetases (E). EPRS, glutamyl-prolyl-tRNA synthetase; DRS, aspartyl-tRNA synthetase; LRS, leucyl-tRNA synthetase; MRS, methionyl-tRNA synthetase. The data are presented as the mean ± SD. ***P < 0.001 by 1-way ANOVA with Dunnett tests (D)., The data shown represent 3 different observations. See also Supplemental Figure 6.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Binding Assay, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A–C) A high-content screening (HCS) platform was processed for migration of M2 macrophages toward KRS-positive (shControl) cells for 7 days (A), migration of KRS-positive cells toward M2 macrophages for 5 or 10 days (B), and migration of M2 macrophages or M1 macrophages that were treated with nothing (–), GAS6, GAS6 + IL8, or GAS6 + IL-8 + ANG toward KRS-positive (shControl) cells and toward KRS-suppressed (shKRS#2) cells for 10 days (C). (D) Coculture of KRS-positive (green) and KRS-suppressed (red) colon cancer cells in spheroids was done before time-lapse imaging for 2 days to observe invasive outgrowth upon treatment with CM from M2 macrophages. Data shown represent 3 independent experiments. Scale bars: 40 μm.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: High Content Screening, Migration, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A) Combined gene expression analysis of KRS and either GAS6 or FGFR1 from public data (GSE28814) for metastasis-free survival or relapse-free survival. (B) Clinical colon cancer patient tissues were immunostained for the indicated molecules. fData shown represent 3 independent experiments.Original magnification, ×40.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: (A) Normal human colon fibroblast (CCD-18Co) cells in 3D collagen I gels were analyzed for laminin expression (green) following treatment with CM of THP-1 monocytes or M1 or M2 macrophages. Nuclei were stained with DAPI (blue). (B–D) CCD-18Co fibroblasts were treated with vehicle, M2 macrophage-CM, or M1 macrophage-CM that had been treated with vehicle or CM from HCT116 spheroids (shControl, shKRS#2, or KRS-WT) for 4 days. Immunostaining was done for laminin (green), and nuclei were stained with DAPI (blue) (B). Laminin α1 (LAMA1), laminin β1 (LAMB1), or fibronectin (FN1) mRNA levels (C) or collagen contraction (D) were analyzed. (E) Collagen gel contraction was analyzed for CCD-18Co fibroblasts in normal media, CM from M2 macrophages, or CM from M1 macrophages that had been treated with GAS6 (G), GAS6 + IL-8 (GI), or GAS6 + IL-8 + ANG (GIA) for 4 days. (F) KRS-positive (shCon, shControl) colon cancer spheroids (green) were cocultured with CCD-18Co fibroblasts (red) that were treated with or without M2 macrophage-CM, before imaging of disseminative outgrowth of cancer cells and fibroblasts. The data are presented as the mean ± SD. *P < 0.05, ***P < 0.001 by 1-way ANOVA with Dunnett tests (C–E). The data shown represent 3 independent experiments.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Expressing, Staining, Immunostaining, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

doi: 10.1172/JCI99806

Figure Lengend Snippet: KRS-positive cancer spheroids induce GAS6/IL-8/ANG for M2 macrophage polarization, and then M2 macrophages secrete FGF2/GROα/M-CSF for signaling activation in cancer spheroids for invasive outgrowth or dissemination. MSC, multi–tRNA synthetase complex.

Article Snippet: The Human XL Cytokine Array Kit (ARY022B, R&D Systems), Multiplex Human Cytokine ELISA Kit (EM10002, ANOGEN), Arginase Activity Assay Kit (ab180877, Abcam), and Human GAS6 ELISA Kit (E-EL-H0078, Elabscience) were also used to simultaneously detect differences in cytokine secretion profiles in the CM of THP-1 or human macrophages (monocyte, M1-polarized, M2-polarized) cells, following the manufacturers’ protocols.

Techniques: Activation Assay

Journal: Cell Death and Differentiation

Article Title: KPNB1-mediated nuclear translocation of PD-L1 promotes non-small cell lung cancer cell proliferation via the Gas6/MerTK signaling pathway

doi: 10.1038/s41418-020-00651-5

Figure Lengend Snippet: a , b Western blot assay of PD-L1, p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 expression in stable PD-L1 knockdown or overexpressed cells when compared to control cells. c , d PD-L1-overexpressed and control cells were treated with 3 μM UNC2025 or 10 μM LDC1267 for 48 h, then the cell lysates were proceed with western blot assay to test PD-L1, p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 protein levels. e Western blot assay of the above proteins in PD-L1 overexpressing cells after knocked down with two separate si-MerTKs. f Western blot assay of p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 protein levels in PD-L1 stable knockdown cells after stimulated with 500 ng/ml rh-Gas6 for 24 h.

Article Snippet: Gas6 level was measured with a human Gas6 ELISA kit (SEA204Hu, USCN Life Sciences, Wuhan, China).

Techniques: Western Blot, Expressing, Knockdown, Control

Journal: Cell Death and Differentiation

Article Title: KPNB1-mediated nuclear translocation of PD-L1 promotes non-small cell lung cancer cell proliferation via the Gas6/MerTK signaling pathway

doi: 10.1038/s41418-020-00651-5

Figure Lengend Snippet: a , b qRT-PCR analysis of Gas6 and PROS1 mRNA expression levels in stable PD-L1 knockdown and overexpression cell lines. c The basal secretion level of Gas6 in PD-L1 stable cell lines and control cells. Data were presented as the mean ± SD. Data were analysed using non-paired Student’s t test, one-way ANOVA analysis followed by Bonferroni’s post hoc test. ** P < 0.01; *** P < 0.001 vs. control or as indicated.

Article Snippet: Gas6 level was measured with a human Gas6 ELISA kit (SEA204Hu, USCN Life Sciences, Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Stable Transfection, Control

Journal: Cell Death and Differentiation

Article Title: KPNB1-mediated nuclear translocation of PD-L1 promotes non-small cell lung cancer cell proliferation via the Gas6/MerTK signaling pathway

doi: 10.1038/s41418-020-00651-5

Figure Lengend Snippet: a , b The results of the luciferase reporter assay in H1299-PD-L1-overexpressed cells and control cells after transfection with a set of luciferase reporter plasmids containing different regions of the Gas6 promoter or mutants at three SP1 binding sites for 24 h. c ChIP assays were performed to detect PD-L1 and Sp1 binding to the Gas6 promoter in H1299-PD-L1-overexpressed cells and control cells. d , e The mRNA and protein levels of Gas6 in H1299 and HCC827 cells after transient transfection with Sp1 siRNAs or Sp1-overexpressing plasmids. f The luciferase activity of the Gas6 promoter in 293T cells transfected with control vector, PD-L1 vector, Sp1 vector or both PD-L1 and Sp1 vectors. g H1299- and HCC827-PD-L1-overexpressed cells were silenced with si-Sp1 or si-NC (control). Data were presented as the mean ± SD. Data were analysed using non-paired Student’s t test, one-way or two-way ANOVA analysis followed by Bonferroni’s post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control or as indicated.

Article Snippet: Gas6 level was measured with a human Gas6 ELISA kit (SEA204Hu, USCN Life Sciences, Wuhan, China).

Techniques: Luciferase, Reporter Assay, Control, Transfection, Binding Assay, Activity Assay, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: KPNB1-mediated nuclear translocation of PD-L1 promotes non-small cell lung cancer cell proliferation via the Gas6/MerTK signaling pathway

doi: 10.1038/s41418-020-00651-5

Figure Lengend Snippet: a EdU assay was performed in H1299 and HCC827 cells transfected with si-MerTK or si-NC with or without Gas6 stimulation for 24 h (Scale bar: 2 mm). b Western blot assay of p-MerTK, MerTK, p-AKT, AKT, p-Erk and Erk. c The proposed mechanistic model underlying the nPD-L1-mediated Gas6/MerTK signaling stimulating NSCLC cell proliferation. Data were presented as the mean ± SD. Data were analysed using one-way ANOVA analysis followed by Bonferroni’s post hoc test. ** P < 0.01; *** P < 0.001 vs. control or as indicated.

Article Snippet: Gas6 level was measured with a human Gas6 ELISA kit (SEA204Hu, USCN Life Sciences, Wuhan, China).

Techniques: EdU Assay, Transfection, Western Blot, Control