Journal: EMBO Molecular Medicine

Article Title: Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma

doi: 10.1002/emmm.201100172

Figure Lengend Snippet: Functional siRNA screen of potential RORγ target genes identified by microarray analysis of 3T3-L1 cells overexpressing RORγ. 3T3-L1 cells were transfected with siRNA pools, differentiated and fluorescently stained to assess the percentage of differentiated cells ( n = 4). Nuclear run-on assay for transcriptional activity of genes in undifferentiated 3T3-L1 cells infected with control or RORγ-overexpressing lentivirus. 32P-labelled mRNA was hybridized to cDNA from the indicated genes on a slot blot and analysed by autoradiography. Expression of Mmp3 mRNA in 3T3-L1 preadipocytes overexpressing RORγ by transient transfection or viral infection. MMP3 promoter constructs with potential ROR response elements (RREs) cloned into a luciferase reporter vector. Luciferase assay in 3T3-L1 preadipocytes overexpressing RORγ or control vector 48 h after transfection normalized to renilla luciferase activity. Activation of IL17 promoter was used as a control ( n = 4). Chromatin immunoprecipitation of RORγ in 3T3-L1 cells using anti-HA antibody 6 days after infection with RORγ or control lentivirus. DNA was quantified by qPCR using specific primers for RRE1 and RRE2 and normalized to input ( n = 3). Fluorescent staining of differentiated 3T3-L1 adipocytes expressing RORγ or control vector during differentiation treated with or without 20 µM of MMP3 inhibitor 2 days before and after induction of differentiation (scale: 100 µm). The results shown are representative of three independent experiments. Values represent means ± SD, * p ≤ 0.05 and ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: mRNA was isolated and transcribed into cDNA using the Multi-MACS cDNA kit (Miltenyi). mRNA expression was assessed by real-time PCR using SybrGreen (Invitrogen) according to the manufacturer's protocol. mRNA expression was normalized to 36b4 or Gapdh.

Techniques: Functional Assay, Microarray, Transfection, Staining, Nuclear Run-on Assay, Activity Assay, Infection, Control, Dot Blot, Autoradiography, Expressing, Construct, Clone Assay, Luciferase, Plasmid Preparation, Activation Assay, Chromatin Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of platelet-derived growth factor in the vascular walls of patients with lower extremity arterial occlusive disease

doi: 10.3892/etm.2015.2275

Figure Lengend Snippet: Reagents used in the study.

Article Snippet: Multi-functional DNA Gel Extraction kit II (Spin-Column) , BioTeke Corporation, Beijing, China.

Techniques: RNA Extraction, Gel Extraction

Journal: Cell reports

Article Title: Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity

doi: 10.1016/j.celrep.2020.108180

Figure Lengend Snippet: (A) Experimental workflow for analysis of freshly isolated (day 0) and stimulated pDCs. Bona fide pDCs (AXL − ) were sorted and analyzed immediately (day 0) or stimulated in vitro for 2 days, followed by re-sorting live cells for ATAC-seq analysis (see and for gating strategy). (B) Left: protein levels of HLA-DR and CD80 in freshly isolated (day 0) or 2-day stimulated bona fide pDCs measured by flow cytometry (n = 3–9 in 3–8 experiments). The statistics are determined by Kruskal-Wallis with Dunn’s multiple comparisons test. Right: IFN-α measured by ELISA in culture supernatant after 2 days (n = 5 in 5 experiments). The statistics are determined by Mann-Whitney test. (C) Genome tracks from 1 representative donor. (D) Left: PCA based on ATAC-seq signal in all cis -elements. Each point represents 1 sample (n = 3–4 per condition). Right: scatterplot comparing all cis -elements between CD40L- and IMIQ-stimulated pDCs. The colored points indicate significantly differentially accessible cis -elements (FC > 2 and p-adj < 0.05). (E) Heatmap of scaled ATAC-seq signal in cis -elements identified in (D). (F) Genome tracks from 1 representative donor. (G) Left: PCA of sorted bona fide pDCs analyzed by CyTOF, including three time points (days 0, 2, and 6) and conditions (media alone, CD40L, IMIQ) subsampled and merged. The color indicates the branch cluster determined by Wishbone (n = 1 representative of 2 experiments). Center: percentage of pDCs in each Wishbone branch at day 6. Right: PCA colored by expression of select markers. (H) Top Gene Ontology terms enriched in CD40L and IMIQ differentially accessible cis -elements. The bubble size represents the fold enrichment. The color indicates −log 10 false discovery rate (FDR). (I) Cytokines in culture supernatant of 2-day stimulated pDCs (n = 5 in 5 experiments). The statistics are determined by t test. (J) Left: frequency of Ki67 + cells among fresh (day 0) or 2-day stimulated pDCs. Right: number of CellTrace Violet low (CTV lo ) cells among fresh, 2-, or 6-day stimulated pDCs (n = 4–11 in 2–7 experiments). The statistics are determined by t test. (K) MLR using fresh or 2-day stimulated pDCs (DC:T cell ratio 1:20, n = 3–4 donors in 3 experiments). The statistics are determined by 1-way ANOVA against day 0 or t test. Bar graphs shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also and .

Article Snippet: Human IFN Alpha Multi-Subtype ELISA Kit (TCM) , PBL Assay Science , Cat# 41105-1.

Techniques: Isolation, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Expressing

Journal: Cell reports

Article Title: Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity

doi: 10.1016/j.celrep.2020.108180

Figure Lengend Snippet: (A) Wanderlust trajectory of fresh (day 0), 2-, or 6-day CD40L-stimulated bona fide pDCs analyzed by CyTOF; each point represents 1 cell (1 experiment of 3–4). (B) Normalized expression of pDC and cDC markers plotted along Wanderlust trajectory axis. (C) As in (A), but colored by expression of key markers. (D) Statistical Scaffold maps of CyTOF data from fresh (day 0), 2-, or 6-day CD40L-stimulated pDCs (1 representative donor). (E) Summary graph of frequency of pDCs mapped to each landmark node (n = 2–3 donors in 3–4 experiments). (F) Protein expression in fresh (day 0), 2-, or 6-day CD40L-stimulated bona fide pDCs analyzed by flow cytometry and CyTOF (n = 3–18 donors in 3–16 experiments). Statistics determined by Kruskal-Wallis with Dunn’s multiple comparisons test. (G) Functional analysis of pDCs that mapped to each landmark node. Two-day CD40L-stimulated pDCs were re-sorted based on phenotype. Left: IFN-α in culture supernatant after 24 h CpG-A, measured by ELISA. Right: T cell proliferation in MLR (n = 2–3 donors in 2–3 experiments). Bar graphs show means ± SDs. **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also and .

Article Snippet: Human IFN Alpha Multi-Subtype ELISA Kit (TCM) , PBL Assay Science , Cat# 41105-1.

Techniques: Expressing, Flow Cytometry, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity

doi: 10.1016/j.celrep.2020.108180

Figure Lengend Snippet:

Article Snippet: Human IFN Alpha Multi-Subtype ELISA Kit (TCM) , PBL Assay Science , Cat# 41105-1.

Techniques: Purification, Recombinant, Produced, SYBR Green Assay, Saline, Lysis, Electron Microscopy, Labeling, Staining, Cell Isolation, Enzyme-linked Immunosorbent Assay, Control, Antibody Labeling, Reverse Transcription, DNA Library Preparation, Generated, Microarray, Software