Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Microarray, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

doi: 10.3389/fimmu.2018.00080

Figure Lengend Snippet: Type I and II interferon genes have time-dependent IL-1B and IL-36 responses. (A) Interferon gamma receptor 1 ( IFNGR1 ). (B) Interferon gamma receptor 2 ( IFNGR2 ). (C) Interferon alpha and beta receptor subunit 2 ( IFNAR2 ). In panels (A–C) , average FPKM (±1 SE) is shown for each group, and asterisks denote significant differences relative to the control (CTL) treatment at the corresponding time point (paired two-sample t -test; n = 2 or 3 per treatment). (D) Genes with interferon response factor 1 (IRF1) binding sites (5 kb upstream region). The middle 50% of FC estimates is shown for genes with 2+ IRF1 binding sites compared with genes with fewer IRF1 sites (magenta font, horizontal axis: P < 0.05, Wilcoxon rank sum test). The IRF1 position weight matrix is shown (right) along with the IRF1 tetrameric structure (bottom right; NCBI structure database). (E) IFN-induced gene signature scores. IFN-induced genes were identified from microarray studies of IFN-treated keratinocytes (left margin), and the average FC for these genes was calculated in IL-1B/IL-36 experiments (bottom margin). Left margin labels indicate the cytokine concentration (in ml), treatment duration, and GEO series accession number. All cytokine experiments were replicated with at least two samples per treatment. (F) Top 30 IFN-g-induced genes (identified from GSE36287). (G) Top 35 INFa-induced genes (identified from GSE36287). (H) Self-organizing maps (SOMs). The SOM layout was determined only from IFN-g-induced genes (i.e., 2,500 genes most strongly induced by IFN-g, GSE36287). Colors reflect average FC estimates for IFN-g-induced genes assigned to each SOM region (columns 1 and 2 on left). The final column (yellow–blue) displays the mean FC difference for each cytokine with respect to each SOM region (8 h mean FC–24 h mean FC; log 2 scale).

Article Snippet: MYD88-KO KCs including WT KCs were grown in 12-well plates, and cells were treated with recombinant IL-1 beta (10 μg/ml; R&D Systems # 201-LB-025), IL-36 gamma (10 μg/ml; R&D Systems # 6835-IL-010), IFN-gamma (50 μg/ml; R&D Systems # 285-IF-100), IL-17A (20 μg/ml; R&D Systems # 317-ILB-050), and/or TNF-alpha (10 μg/ml; R&D Systems # 210-TA-005) for 8 or 24 h. RNAs were isolated from cell cultures using Qiagen RNeasy plus kit (Cat # 74136).

Techniques: Control, Binding Assay, Microarray, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Upregulation of CXCL1 in DCs isolated from mice and patients with CRC. Increased CXCL1, CXCL2 and CXCL3 ( A ) in SW620-conditioned TADCs, as determined by microarray. Elevated CXCL1 in SW620-conditioned TADCs at mRNA ( B ) and protein ( C ) levels. TADCs were generated by culturing CD14 + monocytes with RPMI, L-15 medium (50%), and SW620-CM (50%) and presenting in GM-CSF (10 ng/mL) and IL-4 (10 ng/mL) for five days. The expressions of mRNA and protein were assessed by microarray, qRT-PCR and Luminex assays. ( D ) The level of CXCL1 in the DCs isolated from patients with CRC. CD11c + cells were isolated from healthy donors and patients with CRC, and the levels of CXCL1 were measured by Luminex assay. ( E ) The levels of CXCL1 in DCs isolated from colon cancer-bearing mice. Mouse colon cancer CT26 cells were injected into mice via intraperitoneal injection. After 14 days, peritoneal lymph nodes were harvested. CD11c + DCs were isolated from the lymph nodes, and the culture medium collected after 24 h incubation. CXCL1 levels were determined by ELISA. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Isolation, Microarray, Generated, Quantitative RT-PCR, Luminex, Injection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 increased cancer stem cell (CSC) properties in SW620 colon cancer cells. Anchorage-independent growth ( A ) and tumor spheroid formation ( B ) of CXCL1-treated SW620 cells. The expression of CD44, CD326 and CD133 ( C ) and aldehyde dehydrogenase (ALDH) activity ( D ) in tumor spheroids. SW620 cells were cultured with CXCL1 protein in soft agar (0.4%) or ultra-low attachment wells for 40 and 10 days, respectively. The tumor spheroids were counted by microscope. ALDH activity and surface markers of tumor spheroids were determined by ALDEFLUOR assay and flow cytometry. ( E ) Level of CSC-related transcription factors in tumor spheroids. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 enhanced cancer progression in SW620 cells. CXCL1 increased cell migration, as determined by wound healing ( A ) and transwell ( B ) analysis. The migration ability of SW620 cancer cells was assessed by wound healing assay and transwell assay. CXCL1 acted as a chemoattractant for cancer migration in the transwell system for 24 h. The effect of CXCL1 on the expression of MMP-7 ( C ), EMMPRIN ( D ) and epithelial-to-mesenchymal transition (EMT) ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the levels of various MMPs and EMT markers were determined by Luminex assay and Western blot. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Luminex, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Gene Profile of CXCL1-Treated SW620 Cells.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The correlation of CXCL1-regulated genes with CRC patient outcome. The correlation of overall survival ( A ), relapse-free ( B ) and metastasis-free rates ( C ) with CXCL-1 regulated genes. ( D ) The effect of CXCL1 in the expression of PTHLH and TCF4 at mRNA levels. CXCL1 increased the expression of PTHrP ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the expressions of PTHLH and TCF4 were determined by qRT-PCR and ELISA, respectively. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The effect of CXCL1 on the expression of microRNAs (miRNAs) in colon cancer. ( A ) The regulatory effect of CXCL1 on the expressions of miRNAs in colon cancer. Prognostic significance of miR-105 and miR-597 in overall survival ( B ), relapse-free ( C ) and metastasis-free rates ( D ) in colon cancer patients. The effect of CXCL1 on the levels of miR-105 ( E ) and miR-597 ( F ) in colon cancer. SW620 cells were treated with CXCL1 for 24 h, and the expression of miR-105 and miR-597 determined by qRT-PCR. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*). Hsa, Homo sapiens; TCGA, The Cancer Genome Atlas; HR, hazard ratio; PVAL, p -value.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Proposed model of TADCs in colon cancer. Soluble factors in colon cancer cause abnormal phenotypes of dendritic cells, which in turn increase cancer stem cell properties, cell mobility, and EMT of colon cancer by producing the inflammatory chemokine CXCL1. Transcriptome analysis indicates that CXCL1 increases potential oncogene expression in colon cancer, including PTHLH , TYRP1 , FOXO1 , TCF4 , ZNF880 and the onco-miR miR-105. Our study indicates a new mechanism by which the colon cancer milieu exploits DC plasticity to support cancer progression.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Frontiers in Microbiology

Article Title: Novel Insights Into Cellular Changes in HPV8-E7 Positive Keratinocytes: A Transcriptomic and Proteomic Analysis

doi: 10.3389/fmicb.2021.672201

Figure Lengend Snippet: Transcriptional changes in HPV8-E7 positive keratinocytes. (A) Venn Diagram comparison of two independent cDNA microarray analyses carried out with PHAK expressing HPV8-E7 in the Akgül lab in Cologne and with PHFK expressing HPV8-E7 in the Alonso lab at the DKFZ in Heidelberg. (B) Gene IDs of 4 genes differentially expressed in both array studies. (C,D) GDF15 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (E) GDF15 ELISA performed with cell culture supernatants of HPV8-E7 expressing keratinocytes grown in KGM-Gold medium. (F,G) ATF3 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (H) GADD34 mRNA expression in PM1 keratinocytes cultured in KGM-Gold. RTqPCRs were normalized to HPRT1 mRNA levels ( n = 3 independent experiments performed in duplicate). Error bars represent standard deviations. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control conditions (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The Human GDF15 Quantikine ELISA Kit (RnD systems) was used to measure GDF15 in cell culture supernatants.

Techniques: Comparison, Microarray, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Pathology

Article Title: Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

doi: 10.1002/path.4439

Figure Lengend Snippet: Double minute chromosome (DM)-containing tumour cells often show constitutive activation of the ERK1/2–MAPK pathway. (A, F) The number of DMs/cell in various human malignant tumour cells (bars represent mean ± SD). (B–D, G) Phosphorylation status of ERK1/2, p38 and JNK1/2/3 in DM-containing tumour cells; phosphorylation of ERK1/2, p38 and JNK1/2/3 in MEKK3- or p38-over-expressing or TNF α -treated HEK293T cells (10 ng/ml for 30 min) are shown as positive controls. (E) Microarray analysis of UACC-1598DM compared to UACC-1598HSR cells

Article Snippet: Recombinant human TNF α was purchased from BioVision (Mountain View, CA, USA).

Techniques: Activation Assay, Expressing, Microarray

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: RA enhances wound healing and represses inflammatory responses. ( A ) In vivo wound healing assay. The wounds were created and treated with a control or 0.1% Tretinoin cream for 5 days. The size of wound was monitored daily by recording wound closure (%) (n = 6 per each group) (left). qRT-PCR analyses of Arg1 mRNA at wound sites (right). Each wound tissue was collected at day 5 for mRNA extraction. ( B ) qRT-PCR analyses of LPS-induced (100 ng/ml, 1 h) pro-inflammatory cytokine gene ( TNF-α and IL-6 ) mRNA in RAW264.7 cells treated with conditioned medium (CM, 4 h) collected from IL-4, RA or IL-4/RA-treated (6 h) RAW264.7 cells. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats (mean ± s.d), and Student's t -test (n = 3) was used (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: In Vivo, Wound Healing Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: RA promotes anti-inflammatory macrophage activation by synergizing with IL-4 to activate Arg1 expression. ( A ) qRT-PCR for genes selected from microarray analysis of RAW264.7 cells treated with IL-4, RA or IL-4/RA for 3 h. ( B ) qRT-PCR analyses of Arg1 mRNA in mouse peritoneal macrophages stimulated with IL-4, RA or IL-4/RA for 3 h. ( C ) Western blot analyses of Arg1 protein from the extract of RAW264.7 cells treated with IL-4, RA or IL-4/RA for 24 h. ( D ) qRT-PCR analyses of Raldh2 mRNA in mouse bone marrow-derived macrophages treated with indicated ligands and cytokines for 6 h. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats (mean ± s.d), and Student's t -test (n = 3) was used (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Microarray, Western Blot, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: STAT6 and RAR are required for the synergistic effect of IL-4 and RA on Arg1 activation. ( A–C ) qRT-PCR analyses of ( A ) Arg1 mRNA expression in RAW264.7 cells treated with IL-4, RA or IL-4/RA for 6 h with or without AGN (RAR pan-antagonist, 300 nM) pre-treatment for 30 min ( B ) Control knockdown (Ctrl KD) or STAT6 knockdown (STAT6 KD) cells treated with IL-4, RA or IL-4/RA for 6 h ( C ) Control, STAT6 KD or AGN-pretreated STAT6 KD cells treated with IL-4, RA or IL-4/RA for 6 h. ( D ) qRT-PCR analyses of RAR isoforms mRNA expression in RAW264.7 cells treated with IL-4, RA or IL-4/RA for 6 h. ( E ) Arg1 gene luciferase reporter assay deleted in enhancer (STAT6-binding sites, −2.7 kb), RARE (RAR-response element, −0.8 kb) or both in RAW264.7 cells treated with IL-4, RA or IL-4/RA 24 h. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats (mean ± s.d), and Student's t -test (n = 3) was used (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: STAT6 and RARβ synergistically recruit Med25 upon IL-4 and RA treatment to enhance Arg1 transcription. ( A ) ChIP (Chromatin immunoprecipitation) assay of STAT6 and RARβ in RAW264.7 cells treated with IL-4, RA or IL-4/RA for 30 min. STAT6 and RARβ enrichment on enhancer and RARE, respectively, was measured by qPCR. ( B ) ChIP assay of Med25 on Arg1 enhancer, RARE or +1 nucleosome region in IL-4, RA or IL-4/RA-treated RAW264.7 cells. ( P value: compared with ctrl or single treatment) ( C ) ChIP assay of RARβ or Med25 on RARE or +1 nucleosome, respectively, upon IL-4 and RA treatment with or without AGN (300 nM) pre-treatment for 30 min (left). ChIP assay of STAT6 or Med25 on the enhancer in Ctrl KD or STAT6 KD RAW264.7 cells upon IL-4 and RA treatment (right). ( D ) qRT-PCR analyses of Arg1 mRNA expression in Ctrl KD ( GFP KD) or Med25 KD RAW264.7 cells upon IL-4, RA or IL-4/RA treatment. ( E ) Re-ChIP assay of FLAG-MED25 and STAT6 on the enhancer and the +1 nucleosome of Arg1 at indicated time-points after IL-4 and RA treatment. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats (mean ± s.d), and Student's t -test (n = 3) was used (* P < 0.05; ** P < 0.01; *** P < 0.001). See also Supplementary Figure S3.

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: Med25 mediates synergistic activation of Arg1 by IL-4 and RA via remodeling +1 nucleosome. ( A ) Nucleosome scanning on the Arg1 promoter, the +1 nucleosome and the gene body region in IL-4, RA or IL-4/RA-treated RAW264.7 cells (30 min). ( B ) The +1 nucleosome occupancy immediately down-stream of Arg1 gene's TSS in RAW264.7 cells treated with IL-4, RA or IL-4/RA. ( C ) The +1 nucleosome occupancy in RAW264.7 cells with (upper right) or without (upper left) AGN (300 nM) pre-treatment for 30 min, and that in Ctrl KD (bottom left) or Med25 KD (bottom right) RAW264.7 cells. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats.

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: Activation Assay

Journal: Nucleic Acids Research

Article Title: Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

doi: 10.1093/nar/gkw392

Figure Lengend Snippet: IL-4 and RA synergistically induce Med25 recruitment for transcription initiation-elongation coupling on Arg1 gene. ( A ) ChIP assay of Brg1, RNA Pol II, H3.3, H3K36me3 and TFIIS on the +1 nucleosome in Ctrl KD or Med25 KD RAW264.7 cells with or without IL-4 and RA treatment for 30 min. ( B ) The kinetics of Arg1 pre-mature mRNA synthesis. Total RNAs were isolated to monitor Arg1 pre-mRNA level determined with primers spanning intron 7 and exon 8 regions in qRT-PCR (left). Pre-mRNA synthesizing rate was deduced through measuring the differences in relative ratios of synthesized pre-mRNA per min (right) ( C ) In vitro arginase activity assayed in Ctrl KD or Med25 KD RAW264.7 cells following IL-4, RA or IL-4/RA treatment for 24 h. Cells were treated with IL-4 (10 ng/ml) and/or RA (100 nM) in this figure. Data are representative of three experimental repeats (mean ± s.d), and Student's t -test (n = 3) was used (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Reagent sources: all-trans RA (Sigma, R-2625), 0.1% Tretinoin cream (Boynton Health Service in University of Minnesota), IL-4 (Cell signaling #5208), AGN 193109 (pan-RAR antagonist, Santa Cruz CAS 171746-21-7), LPS (lipopolysaccharide, Sigma, L4391).

Techniques: Isolation, Quantitative RT-PCR, Synthesized, In Vitro, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal:

Article Title: pp32 Reduction Induces Differentiation of TSU-Pr1 Cells

doi:

Figure Lengend Snippet: Expression of IL-6 in TSU-Pr1 cells. TSU-Pr1 cells stably transfected with pp32 anti-sense express higher levels of IL-6 message as compared to parental TSU-Pr1 cells and vector-only control by RT-PCR analysis, which validates the cDNA microarray analysis (see Figure 6).

Article Snippet: Microarray Analysis of TSU-Pr1 Cell Lines This procedure was performed at The Johns Hopkins University Oncology Microarray facility by using a human glass 12K cDNA chip.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Microarray