Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: IL-6 and TNF-α have distinct effects on FLS and chondrocytes. ( a and b ) Mouse FLS ( a ) and chondrocytes ( b ) were infected with the indicated MOI of Ad- Epas1 , and the levels of secreted IL-6 and TNF-α in the culture media were determined by ELISA. ( c and d ) The indicated amounts of recombinant mouse IL-6 protein were used to treat FLS ( c ) or chondrocytes ( d ) for 24 h, and the mRNA levels of Mmp3 and Mmp13 were detected by qRT-PCR. ( e and f) Mouse FLS ( e ) and articular chondrocytes ( f ) were treated with the indicated doses of recombinant mouse TNF-α protein for 24 h, and the levels of the indicated catabolic factors were determined by qRT-PCR. Values are presented as means±s.e.m. (* P <0.01, ** P <0.005).

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: Blockade of TNF-α secretion in co-cultured FLS or chondrocytes. Mouse chondrocytes or FLS were infected with or without 800 MOI of Ad-C or Ad- Epas1 and incubated with FLS ( a ) or chondrocytes ( b ), respectively. After 12 h, the indicated amounts (μg) of TNF-α-neutralizing antibody were added. After an additional 12 h, the transcripts of the indicated catabolic factors were detected by RT-PCR and qRT-PCR. Values are presented as means±s.e.m. (* P <0.05, ** P <0.005).

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Cell Culture, Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: The receptors for IL-6 and TNF-α show distinct expression patterns in HIF-2α-overexpressing FLS and chondrocytes. ( a and b ) Mouse FLS or chondrocytes were infected with Ad- Epas1 or empty virus at an MOI of 800 for 24 h, and microarray analyses were performed on chondrocytes and FLS. Microarray data for IL-6 receptors ( Il6ra and Il6st ) and TNF-α receptors ( Tnfrsf1a and Tnfrsf1b ) in Ad- Epas1 -infected FLS ( a ) and chondrocytes ( b ) are shown. Transcript expression levels are shown relative to those in Ad-C-treated cells. ( c) Schematic diagram summarizing the regulatory mechanism governing the in vitro interaction between FLS and chondrocytes in the presence of HIF-2α overexpression.

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Expressing, Infection, Virus, Microarray, In Vitro, Over Expression

Journal: Malaria Journal

Article Title: Differential gene expression mediated by 15-hydroxyeicosatetraenoic acid in LPS-stimulated RAW 264.7 cells

doi: 10.1186/1475-2875-8-195

Figure Lengend Snippet: Select Gene Expression Changes Mediated by 15-HETE a

Article Snippet: Following RT, all assays were performed with Applied Biosystems TaqMan FAM labeled 20× probes: Arf3 (Taqman assay Mm00500194_m1), Cldn11 (Mm00500915_m1), Cxcl11 (Mm00444662_m1), Mapk14 (Mm00442497_m1), Prdx1 (Mm01621996_s1), Sdc1 (Mm00448918_m1), and Egr1 (Mm00656724_m1).

Techniques: Gene Expression, Binding Assay, Ubiquitin Proteomics, Virus, Transduction, Membrane, Activation Assay

Journal: Malaria Journal

Article Title: Differential gene expression mediated by 15-hydroxyeicosatetraenoic acid in LPS-stimulated RAW 264.7 cells

doi: 10.1186/1475-2875-8-195

Figure Lengend Snippet: Quantitative real-time RT-PCR validation of microarray results . RAW 264.7 cells were stimulated with 0.1 μg/mL LPS and treated with 40 μM 15-HETE for 24 h prior to RNA extraction. Fold-changes (treated stimulated cells relative to stimulated cells) are shown ( ± 99% confidence interval for quadruplicate measurements of n = 3 biological replicates). Abbreviations: Arf3 (ADP-ribosylation factor 3), Cldn11 (claudin 11), Cxcl11 (chemokine (C-X-C motif) ligand 11), Egr1 (early growth response 1), Mapk14 (mitogen-activated protein kinase 14), Prdx1 (peroxiredoxin 1), and Sdc1 (syndecan 1).

Article Snippet: Following RT, all assays were performed with Applied Biosystems TaqMan FAM labeled 20× probes: Arf3 (Taqman assay Mm00500194_m1), Cldn11 (Mm00500915_m1), Cxcl11 (Mm00444662_m1), Mapk14 (Mm00442497_m1), Prdx1 (Mm01621996_s1), Sdc1 (Mm00448918_m1), and Egr1 (Mm00656724_m1).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Microarray, RNA Extraction

Journal: Human reproduction (Oxford, England)

Article Title: Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle.

doi: 10.1093/humrep/deu008

Figure Lengend Snippet: Figure 2 Validation of microarray results by qRT-PCR in 11 selected genes in 7 paired samples of GC prior to and 36 h after rhCG triggering. All validated genes were significantly differentially expressed before compared with after rhCG in the qRT-PCR experiment (P-values, ratio paired t-test): CD24: P ¼ 0.0069, AREG: P ¼ 0.0015, PTGS2: P ¼ 0.0004, COL6A3: P ¼ 0.0007, HSD11B1: P ¼ 0.0001, HSD11B2: P ¼ 0.0132, INHBA: P ¼ 0.0012, CCNA2: P ¼ 0.0037, PTTG1: P ¼ 0.0049, IGF2: P ¼ 0.0064 and CYP19A1: P ¼ 0.0010. GC, granu- losa cells.

Article Snippet: Validation of differential expression by quantitative reverse transcriptase PCR (qRT-PCR) The following genes were testedby pre-designed TaqManw Gene Expression Assays (gene symbol/catalogue number): CD24 (Hs03044178_g1), AREG (Hs00950669_m1), HSD11B1 (Hs01547870_m1), HSD11B2 (Hs003886 69_m1), PTGS2 (Hs00153133_m1), COL6A3 (Hs00915125_m1), CYP19A1 (Hs00903413_m1), IGF2 (Hs04188276_m1), PTTG1 (Hs00851754_u1), CCNA2 (Hs00996788_m1) and INHBA (Hs01081598_m1) as well as the Endogenous Control Assays for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems/Life Technologies Europe, Nærum, Denmark).

Techniques: Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Evolution & development

Article Title: Abundant genetic variation in transcript level during early Drosophila development

doi: 10.1111/j.1525-142X.2008.00281.x

Figure Lengend Snippet: Maternal and zygotic gene expression

Article Snippet: Found on Affymetrix Microarray Expressed Varying among lines Varying over time Increasing Declining Changes during development 2089 (AST7 * ) 2065 ** (ST4) 1215 (ST5) 950 (ST6) 698 (ST7) 224 (ST8) 491 (ST9) Strictly maternal 27 (AST12) 27 (ST10) 21 (ST11) 21 (ST12) 19 (ST13) 0 19 (ST14) Maternal 49 (AST13) 50 (ST15) 41 (ST16) 35 (ST17) 22 (ST18) 2 (ST19) 20 (ST20) Strictly Zygotic 53 (AST19) 57 (ST27) 29 (ST28) 25 (ST29) 22 (ST30) 19 (ST31) 3 (ST32) Zygotic 532 (AST18) 514 (ST21) 176 (ST22) 141 (ST23) 127 (ST24) 89 (ST25) 38 (ST26) Open in a separate window * AST stands for Supplementary Table in Arbeitman et al. 2002 ; and ST is for the Supplementary Table in this article.

Techniques: Microarray

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: Lactate (Lac) induces the expression of early growth response protein 1 (EGR1) (A) BMDCs from WT mice were stimulated with LPS (100 ng/mL) and/or Lac (20 mM) for 4 hr, and the total RNA from these cells was subjected to microarray analysis. The log2 ratios for the indicated genes in BMDCs stimulated with LPS plus Lac are arranged in descending order. (B) Western blot analysis of EGR1 expression in BMDCs stimulated with LPS (100 ng/mL) and/or Lac (20 mM) for 24 hr. (C) EGR1 expression (green) in RAW 264.7 cells stimulated with Lac (20 mM) or control medium (Ctrl) for 24 hr. Nuclei were stained with Hoechst 33,342 (blue). Scale bars = 10 μm. (D) Tumor tissues or control normal skin (Ctrl) from WT mice at two weeks after B16-F1 cell implantation were examined for EGR1 expression (green). Scale bars = 100 μm. (E) Median fluorescence intensity (MFI) of EGR1 in leukocyte subpopulations in tumor tissues of WT mice 3 weeks post-B16-F1 cell implantation was analyzed using flow cytometry. On days 6, 9 and 12 after injection of B16-F1 cells, Lac (5 mM, 200 μL) or PBS control (200 μL) was intratumorally administered. Data are expressed as mean ± SD. (F) The relationship between EGR1 MFI of tumor-infiltrating DCs and tumor weight (left) or lactate level (right) in the tumor tissues of WT mice 3–4 weeks post-B16-F1 cell implantation. Individual findings are plotted and each data point represents one mouse. Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. Pearson's correlation coefficient was used to evaluate the correlations. ∗ p < 0.05; ∗∗ p < 0.01; n.s., not significant ( p > 0.05). See also and .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Expressing, Microarray, Western Blot, Control, Staining, Fluorescence, Flow Cytometry, Injection, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: EGR1 is expressed in DCs and correlates with LDHA in human melanoma tissues (A) Tumor tissues from patients with melanoma in situ and invasive melanoma were examined by fluorescent staining (Hoechst 33,342, blue), as well as by immunofluorescence analysis for EGR1 (green). Nuclei (enclosed within light blue lines) and EGR1-positive cells (enclosed within magenta lines) were counted automatically using the In Cell Analyzer software. Scale bars = 50 μm. (B) EGR1-positive ratios in tumor tissues of melanoma in situ and invasive melanoma are plotted (see also ). Each data point represents one patient. Bars indicate the mean. (C) Relationship between EGR1-positive ratio and tumor thickness (left) or serum 5-S-cysteinyl-DOPA (5-S-CD) levels (right). Individual findings are plotted and each data point represents one patient. (D) Primary tumor tissues from patients with invasive melanoma were examined by fluorescent staining as indicated. Scale bars = 50 μm. (E) Correlation analysis of EGR1 and LDHA expression levels using RNA-seq data set acquired from primary tumors from 45 skin cutaneous melanoma patients in TCGA (TCGA-SKCM). Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. Pearson's correlation coefficient was used to evaluate the correlations. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: In Situ, Staining, Immunofluorescence, Software, Expressing, RNA Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: CD80 expression level is increased EGR1-deficient DCs (A) Levels of IL-12 p40, TNF-α, and IL-6 secreted by BMDCs stimulated with LPS (100 ng/mL) for 24 hr were measured by ELISA. Data are expressed as mean ± SD from two independent experiments (n = 3–5). (B) RNA from BMDCs from WT and Egr1 −/− mice was subjected to microarray analysis. The log2 ratio was determined for the corresponding genes categorized in cell adhesion molecules within KEGG pathways and the results were arranged in descending order. Genes that overlapped with TLR signaling pathways are in red boxes. (C) Flow cytometric analyses of CD80 and CD86 in CD11c + DCs within splenocytes from WT and Egr1 −/− mice. Representative plots are shown (left). Data are expressed as mean ± SD from two independent experiments (right, n = 3). MFI, median fluorescence intensity. (D) BMDCs from WT or Egr1 −/− mice were stimulated with LPS (100 ng/mL) and/or lactate (Lac, 20 mM) for 24 hr and the MFI of CD80 was assessed (n = 3). Data are expressed as mean ± SD. (E) CFSE-labeled CD8 + T cells from OT-I transgenic mice were co-cultured with BMDCs from WT or Egr1 −/− mice pulsed with Ova 257–264 peptide. Proliferation of OT-I CD8 + T cells was assessed after 72 hr by flow cytometry. (F and G) Tumor growth kinetics in WT or Egr1 −/− mice subcutaneously injected with 1 × 10 6 B16-F1 melanoma cells and treated with anti-PD-L1 antibody or control antibody (Ctrl), Lac and/or PBS as indicated. On days 6, 9, and 12 after injection of B16-F1 cells, 200 μg of control IgG Ab or anti-PD-L1 Ab were injected intraperitoneally (F), and Lac (5 mM, 200 μL) or PBS control (200 μL) was intratumorally administered (G). Data are shown as mean ± SD of 3–4 mice per group. (H) Tumor growth kinetics in diphtheria toxin (DTX)-treated CD11c-DTR bone marrow chimeric mice with subcutaneous injection with 1 × 10 6 B16-F1 melanoma cells and treated intratumorally with 1×10 6 WT or Egr1 −/− BMDCs. Data are shown as mean ± SD (n = 4 mice per group). Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. ∗ p < 0.05; ∗∗ p < 0.01; n.s., not significant ( p > 0.05). See also and .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microarray, Protein-Protein interactions, Fluorescence, Labeling, Transgenic Assay, Cell Culture, Flow Cytometry, Injection, Control, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: EGR1 interacts with SRF and may regulate its activation on the Cd80 promoter (A) ChIP-seq enrichment profiles for H3K27Ac at the Cd80 locus were generated using WT or Egr1 −/− BMDCs stimulated with LPS (100 ng/mL) and lactate (Lac, 20 mM) for 24 hr. (B) ChIP-seq enrichment profiles for EGR1 at the transcription start site (TSS) at the Cd80 locus were generated using WT BMDCs stimulated by LPS (100 ng/mL) and Lac (20 mM) for 24 hr. (C) The 3D protein structure of EGR1 and the EGR1 binding sequence from Protein DataBank were analyzed by CLC Genomics Workbench. (D) SRF was predicted as a potential transcription factor that binds to the region identified in (B) using TFBIND software. SRF was previously reported to interact with EGR1. TF, transcription factor. (E) ChIP-PCR analysis was performed using RAW 264.7 cells overexpressing Srf . ChIP was performed using control IgG (Ctrl) or anti-SRF antibody. PCR was performed using primers that detect sequences in the Cd80 promoter. (F) Co-immunoprecipitation experiments assessing EGR1 binding to SRF. Cell extracts from RAW 264.7 cells overexpressing both Srf and Myc-tagged Egr1 were immunoprecipitated with anti-SRF antibody or control IgG (Ctrl) and analyzed with anti-c-Myc and anti-SRF antibody by Western blotting. (G) Proposed schematic model of how lactate and its induction of EGR1 switches a hot tumor to a cold tumor. Lactate induces a shift from a hot tumor (presence of inflammation) to a cold (non-inflamed) tumor, while TLR4 ligands cause the opposite shift. Lactate upregulates the expression of EGR1, which may downregulate the expression of CD80. See also .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Activation Assay, ChIP-sequencing, Generated, Binding Assay, Sequencing, Software, Control, Immunoprecipitation, Western Blot, Expressing

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet:

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Control, Recombinant, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Reporter Assay, Mutagenesis, Chromatin Immunoprecipitation, DNA Purification, Staining, Lactate Assay, Microarray, Negative Control, Plasmid Preparation, Software, Transfection