Journal: Neuro-Oncology

Article Title: Therapy-induced senescent glioblastoma cells sustain a procancer immune microenvironment by activating DDX58-mediated STAT1 signaling

doi: 10.1093/neuonc/noaf107

Figure Lengend Snippet: DDX58 regulated the protein stability of STAT1 via the ubiquitin E3 ligase TRIM21 ( A ). Western blot analysis of the DDX58, P21, and STAT1 proteins and their phosphorylation in DOX- or TMZ-induced senescent LN229 cells transduced with DDX58-targeting siRNAs. ( B-C ). Western blot analysis of the P21, DDX58, and STAT1 proteins and their phosphorylation in LN229 (B) and U87MG (C) cells overexpressing DDX58. ( D ). Protein stability assays to assess the effect of DDX58 on the STAT1 protein. DDX58-knockdown senescent LN229 cells were treated with cycloheximide (50 μg/mL) for up to 9 h, and STAT1 and DDX58 expression was tested via western blotting. ( E ). LN229 cells were transfected with DDX58 siRNA and then treated with MG132 (20 μM) for 5 h. ( F ). The binding between DDX58 and STAT1 was examined by co-IP and western blotting. ( G ). LN229 cells were transiently transfected with DDX58 siRNA and then treated with TMZ or DMSO, and the changes in the ubiquitin level of STAT1 in LN229 cells were examined by co-IP and western blotting. All the samples were treated with 20 µM MG132 for 2 h. ( H ). Regulators involved in the regulation of STAT1 ubiquitination were screened by transient transfection of LN229 cells with the His-STAT1 pcDNA 4.0 plasmid. One sample was treated with 50 μM TMZ for 4 days. All the cells were examined via co-IP and western blotting ( I ). LN229 cells were transiently transfected with the His-STAT1 plasmid, and changes in the STAT1 binding to TRIM21 in LN229 cells overexpressing DDX58 were examined via co-IP and western blotting. ( J ). Schematic diagram of the DDX58/RIG-I protein domains. P21 and STAT1 protein expression and phosphorylation by overexpressing the CARD, CTD, and helicase domains of DDX58 in LN229 and 293FT cells. ( K ). THP-1 macrophages were cocultured with CM from LN229 cells (CON335, DDX58 overexpressing, DDX58 overexpressing plus fludarabine, 50 ng/mL CSF-1) for 48 h. Scale bars: 1.5 mm, 150 μm. ( L ). Statistical analysis of the data in (K). The samples were analyzed in triplicate with 3 fields per well; **** p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. ( M ). ELISA analysis of CSF1 in LN229 cells overexpressing DDX58 or treated with fludarabine. Comparisons were performed with two-tailed Student’s t tests. * p < 0.01, *** p < 0.001. All the data are presented as the means ± SDs.

Article Snippet: Secreted CSF1/M-CSF protein levels were measured via a human macrophage colony-stimulating factor (M-CSF) ELISA kit (CUSABIO CSB-E04658h).

Techniques: Ubiquitin Proteomics, Western Blot, Phospho-proteomics, Transduction, Knockdown, Expressing, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Niacin Reverses Migratory Macrophage Foam Cell Arrest Mediated by oxLDL In Vitro

doi: 10.1371/journal.pone.0114643

Figure Lengend Snippet: A . Bone marrow derived macrophages were stained with fluorochrome labeled anti-F4/80 and anti-CD36 antibodies. Fluorescence intensity was analysed by flow cytometry. B . Migrated RAW264.7 cells on the lower side of the membrane were stained with DAPI and counted under a fluorescence microscope using a ×10 objective. Representitive pictures of each treatment group. C . Migration of oxLDL treated cells was significantly reduced compared to unstimulated cells. Migration of oxLDL treated cells was significantly different compared to Nicotinic acid 1 mM, Nicotinamide 1 mM and 3 mM. (*, P<0.05, n = 5).

Article Snippet: After 24 hours, 20 ng/ml mouse macrophage colony stimulating factor (M-CSF Mouse, CS-C2038, Cellsystems) was added into medium.

Techniques: Derivative Assay, Staining, Labeling, Fluorescence, Flow Cytometry, Membrane, Microscopy, Migration

Journal: Oncology Letters

Article Title: CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

doi: 10.3892/ol.2017.7420

Figure Lengend Snippet: Effect of silencing CITED2 on macrophage infiltration in vivo and recruitment in vitro . (A) Representative images of immunohistochemical analysis revealed staining for the macrophage marker F4/80, as indicated by 3,3-diaminobenzamindine staining (brown) and visualized using a light microscope (magnification, ×200). The adjacent histograms represent the average number of macrophages/mm 2 . *P<0.05 compared with the scramble group. (B) Representative images revealed macrophage recruitment in response to conditioned media obtained from scramble and shCITED2-expressing breast cancer cells at 6 and 20 h, respectively, using an in vitro Transwell migration assay (magnification, ×400). The adjacent histograms represent quantification of the average number of macrophages recruited at each time point. *P<0.05; **P<0.01 vs. scramble CM. CITED2, Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2; sh, short hairpin; CM, conditioned media.

Article Snippet: Cells were permitted to attach in a 100-mm culture dish overnight at 37°C in a 5% CO 2 incubator with 5 ng/ml macrophage colony stimulating factor (M-CSF; Promega Corporation, Madison, WI, USA).

Techniques: In Vivo, In Vitro, Immunohistochemical staining, Staining, Marker, Light Microscopy, Expressing, Transwell Migration Assay

Journal: Oncology Letters

Article Title: CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

doi: 10.3892/ol.2017.7420

Figure Lengend Snippet: Effect of CITED2 silencing on the expression of macrophage chemotactic factors. mRNA expression in (A) orthotopic xenograft tumors and (B) cells in culture, as determined by reverse transcription-quantitative polymerase chain reaction. (C) Protein expression in the two groups, as determined by ELISA. (D) Localization of CITED2 to the CCL20 promoter, as assessed by a chromatin immunoprecipitation assay using anti-CITED2 or IgG antibodies (control) in MDA-MB-231 and MDA-MB-468 cells. *P<0.05; **P<0.01; ***P<0.001 vs. scramble group (A-C) and vs. IgG (D). CITED2, Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2; sh, short hairpin; CCL, C-C motif chemokine ligand; Ig, immunoglobulin; IL, interleukin; CX3CL, C-X3-C motif chemokine ligand.

Article Snippet: Cells were permitted to attach in a 100-mm culture dish overnight at 37°C in a 5% CO 2 incubator with 5 ng/ml macrophage colony stimulating factor (M-CSF; Promega Corporation, Madison, WI, USA).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Control

Journal: Oncology Letters

Article Title: CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

doi: 10.3892/ol.2017.7420

Figure Lengend Snippet: Effect of inhibiting CCL20 on the induction of macrophage recruitment by MDA-MB-231 cell-conditioned media. Top: Representative images revealed macrophage recruitment by in vitro Transwell migration assay. Bottom: Quantification of the average number of macrophages recruited per experimental condition. ***P<0.001. Magnification, ×400. CCL, C-C motif chemokine ligand; Ig, immunoglobulin; neu, neutral.

Article Snippet: Cells were permitted to attach in a 100-mm culture dish overnight at 37°C in a 5% CO 2 incubator with 5 ng/ml macrophage colony stimulating factor (M-CSF; Promega Corporation, Madison, WI, USA).

Techniques: In Vitro, Transwell Migration Assay