Journal: Frontiers in Immunology

Article Title: Aryl Hydrocarbon Receptor Promotes IL-10 Expression in Inflammatory Macrophages Through Src-STAT3 Signaling Pathway

doi: 10.3389/fimmu.2018.02033

Figure Lengend Snippet: AhR upregulated LPS-induced IL-10 expression in RAW264.7 cells independent of AhR genomic pathway. AhR and Flag protein (A) and mRNA (B) levels were detected in RAW, RAW/NC, and RAW/AhR cells. The experiment was repeated three times; combined results are compared using one-way ANOVA test (RAW/NC, P < 0.001; RAW/AhR, P < 0.001). (C,D) RAW/NC and RAW/AhR cells were stimulated with LPS at the indicated time points. (C) IL-10 mRNA levels were detected with qRT-PCR. The experiment was repeated three times; combined results are compared using independent Student' s t -test ( P = 0.025) (D) Supernatants were collected 12 h after LPS stimulation and IL-10 production was measured with ELISA. The experiment was repeated six times; combined results are compared using one-way ANOVA test ( P = 0.002). (E) Peritoneal macrophages were pre-treated with TCDD (1 nM), β-naphthoflavone (1 μM), CH-223191 (10 μM), indirubin-3′-oxime (1 μM), and benzo[a]pyrene (10 μM), respectively, for 1 h and then stimulated with or without LPS for 12 h. IL-10 expression was measured with ELISA. (F) Schematic of promoter construct of murine AhR gene containing 1054 bp upstream of the transcriptional start site cloned into a luciferase reporter vector. Position of the AhR-binding site are presented spanning −631 to −607 bp and −319 to −343 bp. (G,H) RAW/NC and RAW/AhR cells were stimulated with or without LPS for 2 h and nuclear-extract proteins were then prepared and incubated with AhR-binding site probe 1 (G) and probe 2 (H) of the IL-10 promoter. Binding activity was measured using EMSA. (I) 293T/NC and 293T/AhR cells were co-transfected with a luciferase reporter gene construct of the murine IL-10 promoter and pRL-TK control plasmids for 48 h. The cells were lysed and luciferase activity was analyzed. AhR and Flag protein levels were detected in 293T/NC and 293T/AhR cells (right). Western blot and EMSA images are representative of three independent experiments. Data in all bar graphs are mean ± SEM of three independent experiments. * P < 0.05; NS, not significant.

Article Snippet: RAW264.7 cells (ATCC®-TIB-71TM) and HEK 293T cells (ATCC®-CRL-11268TM) were maintained in RPMI1640 and DMEM medium, respectively, supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FBS.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Construct, Clone Assay, Luciferase, Plasmid Preparation, Binding Assay, Incubation, Activity Assay, Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Integrative Structural Modeling of Intrinsically Disordered Regions in a Human HDAC2 Chromatin Remodeling Complex

doi: 10.1101/2025.08.08.669391

Figure Lengend Snippet: A . Nuclear Localization of HALO-C16orf87 in HEK293T. Halo-tagged proteins are labeled with HaloTag® TMR ligand (red). Nuclei are stained with Hoechst dye (blue). B . Heatmap plotting relative protein abundance expressed as log2FC with the hierarchical bi-clustering of the 8 Halo-tagged baits: C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, SAP30, with the brightest yellow indicating high log2FC (Suppl. Table S1A). C . A network analysis was performed using Cytoscape on members of HDAC1/2 associated complexes significantly enriched with at least one of the bait proteins compared to controls (log2FC > 3, FDRup< 0.05; Suppl. Table S1A). The Halo-tagged bait proteins (C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30) are source nodes in gray, with SIN3A associated proteins in pink, NuRD associated proteins in light blue, CoREST associated proteins in green, MIER associated proteins in purple, HDAC1/2 (in light yellow/orange, and C16orf87 in red. D . Representative images of acceptor and donor fluorescence for each sample in the AP-FRET experiments. SNAP-F-C16orf87 is labeled with the SNAP-Cell® 505-Star ligand, while HDAC1/2-Halo are labeled with the HaloTag® MR ligand (see data in Suppl. Table S2A).

Article Snippet: HEK293T cells (ATCC1CRL-11268TM) were purchased from American Type Culture Collection (ATCC).

Techniques: Labeling, Staining, Quantitative Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Integrative Structural Modeling of Intrinsically Disordered Regions in a Human HDAC2 Chromatin Remodeling Complex

doi: 10.1101/2025.08.08.669391

Figure Lengend Snippet: A. Nuclear localization of SNAP-Flag-C16orf87 in Flp-In TM -293 cells stably expressing HDAC1-Halo or HDAC2-Halo. Halo-tagged proteins are labeled with HaloTag® TMR ligand (red); SNAP-tagged proteins are labeled with SNAP-Cell ® 505-Star ligand (green); nuclei are stained with Hoechst dye (blue). Nuclear localization was also observed with a Halo-tagged C16orf87 construct . B. Bait-specific hierarchical clustering on Topological scores (TopS) including the 8 Halo-tagged baits: C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30. Red corresponds to high TopS values and blue corresponds to negative TopS values (Suppl. Table S1B). C. AP-FRET analysis between SNAP-F-C16orf87 (labeled with SNAP-Cell® 505-Star ligand) and HDAC1/2-Halo (labeled with HaloTag® MR ligand). The positive control was a Halo-NLS-SNAP construct to measure the FRET efficiency of Halo and SNAP when close together; the negative control confirmed an absence of FRET in cells expressing NLS-Halo and NLS-SNAP on their own. Unpaired t-tests were used for statistical analyses where ***: p≤ 0.0001. Representative images of acceptor and donor fluorescence are provided in , while all measurements and Image J processing are reported in Suppl. Table S2A. D. Deacetylase activity assays were performed using equal volumes of eluates from the Halo-only, HDAC2-Halo, and Halo-C16orf87 affinity purifications. Deacetylase activity was measured by a colorimetric reaction, and relative fluorescence unit (RFU) values were normalized to total protein content in each sample as measured by BCA protein assay. Assays were performed in triplicate with error bars representing SEM. Experimental workflow, acquisition setting, raw measurements, and calculations are reported in Supp. Table S2B. E. HEK293T cell lysates expressing tagged C16orf87 with or without 7 Halo-tagged baits (MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30) were used for Halo pulldown analysis. Equal volumes of eluate were analyzed by SDS PAGE and Western blotting. SNAP-FLAG-C16orf87 was detected using anti-FLAG mouse monoclonal primary antibody and IRD 680 LT labeled goat anti-Mouse secondary antibody. Halo-tagged baits were detected using anti-Halo rabbit polyclonal primary antibody and IRD 800CW labeled goat anti-Rabbit secondary antibody. F. Quantification of relative C16orf87 enrichment in HDAC1/2 and MIER protein pulldowns. Normalized values account for differences in C16orf87 concentration between Halo-tagged samples determined by quantitative Western blotting (Suppl. Table S2C). Band intensities were quantitated using Image Studio v5.2 (Li-Cor). Unpaired t-tests were performed for statistical analyses where *** = p≤0.0001.

Article Snippet: HEK293T cells (ATCC1CRL-11268TM) were purchased from American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Labeling, Staining, Construct, Positive Control, Negative Control, Fluorescence, Histone Deacetylase Assay, Activity Assay, Bicinchoninic Acid Protein Assay, SDS Page, Western Blot, Concentration Assay