Journal: Journal of Clinical Investigation

Article Title: The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

doi: 10.1172/jci200522758

Figure Lengend Snippet: Figure 6 Chemokines and chemokine receptors are expressed in human PTC-derived cell lines. (A) CXCL1 and CXCL10 secretion in human PTC cells was evaluated by ELISA. Experiments were performed in triplicate, and the average value of the results ± SD was plotted. Normal thyroid cells (P5) were used as a negative control. (B) Expression levels of CXCR2 and CXCR3 in PTC cell lines were evaluated by Q-RT-PCR. Expression values were calculated relative to the expression level in normal P5 cells. Experiments were performed in triplicate, and the average value of the results ± SD was plotted. (C) Flow cytometric analysis of surface expression of CXCR2 and CXCR3 in TPC1 cells. (D) TPC1 cells were transfected by BRAF or scrambled siRNA and harvested 72 or 96 hours later. Protein lysates were subjected to immunoblotting with anti-BRAF and anti–phospho-p44/p42 ERK antibodies. (E) BRAF siRNA interference in TPC1 cells affected chemokine production as determined by ELISA.

Article Snippet: We measured CXCL1 and CXCL10 levels in culture supernatants using a quantitative immunoassay ELISA kit (QuantiKine ELISA kit; R&D Systems), following the manufacturer’s instructions.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot

Journal: Cell reports

Article Title: Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy

doi: 10.1016/j.celrep.2022.111018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-Thermogenin (UCP1) α-Diagnostics Cat#UCP11-A; RRID:AB_1876090 Mouse monoclonal β-Actin (HRP conjugated) Santa Cruz Cat#sc-47778 HRP; RRID:AB_2714189 Goat anti-rabbit IgG Cell Signaling Cat#7074; RRID:AB_2099233 Guinea pig anti-insulin IgG Dako Cat#IR002; RRID:AB_2800361 Rabbit anti-Ki-67 IgG Invitrogen Cat#MA5-14520; RRID:AB_10979488 Goat anti-guinea pig IgG Jackson ImmunoResearch Cat#106-545-003; RRID:AB_2337438 Goat anti-rabbit IgG Invitrogen Cat#A11036; RRID:AB_10563566 Chemicals, peptides, and recombinant proteins Corticosterone pellets (5mg, 21-day release) Innovative Research of America Cat#G-111 Placebo pellets (5mg, 21-day release) Innovative Research of America Cat#C-111 Corticosterone complexed with 2-hydroxypropyl-β-cyclodextrin Sigma-Aldrich Cat#C174 Glucose Sigma-Aldrich Cat#G8270 Insulin Sigma-Aldrich Cat#I9278 Normal Goat Serum Jackson ImmnuoResearch Cat#005-000-121 SlowFade™ Gold Antifade Mountant with DAPI Thermo Scientific Cat#S36939 Critical commercial assays ELISA corticosterone Arbor Assays Cat#K014-H1 ELISA insulin Crystal Chem Cat#90080 Free Fatty Acid Quantitation Kit Sigma Cat#MAK044 Triglyceride Determination Kit Sigma Cat#TR0100 RNeasy Lipid Tissue Mini Kit Qiagen Cat#74804 RNeasy Mini Kit Qiagen Cat#74106 iScript cDNA Synthesis Bio-Rad Cat#1708890 PowerUp SYBR Green Master Mix ThermoFisher Cat#4368577 RNeasy Lipid Tissue Mini Kit Qiagen Cat#74804 Qscript™ cDNA Synthesis Kit VWR Cat#101414-098 iTaq™ Universal SYBR® Green Supermix Bio-Rad Cat#1725121 L-Type Triglyceride M Enzyme Color A Fujifilm Healthcare Cat#994-02891 L-Type Triglyceride M Enzyme Color B Fujifilm Healthcare Cat#990-02991 Multi-Calibrator Lipid Fujifilm Healthcare Cat#464-01601 Deposited data RNA-seq This paper NCBI Geo GSE202800 Experimental models: Organisms/strains Mice: C57BL/6J Jackson Labs Cat#000664 Mice: C57BL/6J; CD36ko Jackson Labs Cat#019006 Oligonucleotides See Table S8 This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/download.html Fiji software plug-in Adiposoft Galarraga et al. (2012) https://imagej.net/plugins/adiposoft GraphPad Prism 9 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Custom RNAseq analysis codes This paper https://doi.org/10.5281/zenodo.6588146 Open in a separate window KEY RESOURCES TABLE All original code has been deposited on github and is publically available as of the data of publication.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Quantitation Assay, SYBR Green Assay, Software

Journal: Cell reports

Article Title: Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy

doi: 10.1016/j.celrep.2022.111018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-Thermogenin (UCP1) α-Diagnostics Cat#UCP11-A; RRID:AB_1876090 Mouse monoclonal β-Actin (HRP conjugated) Santa Cruz Cat#sc-47778 HRP; RRID:AB_2714189 Goat anti-rabbit IgG Cell Signaling Cat#7074; RRID:AB_2099233 Guinea pig anti-insulin IgG Dako Cat#IR002; RRID:AB_2800361 Rabbit anti-Ki-67 IgG Invitrogen Cat#MA5-14520; RRID:AB_10979488 Goat anti-guinea pig IgG Jackson ImmunoResearch Cat#106-545-003; RRID:AB_2337438 Goat anti-rabbit IgG Invitrogen Cat#A11036; RRID:AB_10563566 Chemicals, peptides, and recombinant proteins Corticosterone pellets (5mg, 21-day release) Innovative Research of America Cat#G-111 Placebo pellets (5mg, 21-day release) Innovative Research of America Cat#C-111 Corticosterone complexed with 2-hydroxypropyl-β-cyclodextrin Sigma-Aldrich Cat#C174 Glucose Sigma-Aldrich Cat#G8270 Insulin Sigma-Aldrich Cat#I9278 Normal Goat Serum Jackson ImmnuoResearch Cat#005-000-121 SlowFade™ Gold Antifade Mountant with DAPI Thermo Scientific Cat#S36939 Critical commercial assays ELISA corticosterone Arbor Assays Cat#K014-H1 ELISA insulin Crystal Chem Cat#90080 Free Fatty Acid Quantitation Kit Sigma Cat#MAK044 Triglyceride Determination Kit Sigma Cat#TR0100 RNeasy Lipid Tissue Mini Kit Qiagen Cat#74804 RNeasy Mini Kit Qiagen Cat#74106 iScript cDNA Synthesis Bio-Rad Cat#1708890 PowerUp SYBR Green Master Mix ThermoFisher Cat#4368577 RNeasy Lipid Tissue Mini Kit Qiagen Cat#74804 Qscript™ cDNA Synthesis Kit VWR Cat#101414-098 iTaq™ Universal SYBR® Green Supermix Bio-Rad Cat#1725121 L-Type Triglyceride M Enzyme Color A Fujifilm Healthcare Cat#994-02891 L-Type Triglyceride M Enzyme Color B Fujifilm Healthcare Cat#990-02991 Multi-Calibrator Lipid Fujifilm Healthcare Cat#464-01601 Deposited data RNA-seq This paper NCBI Geo GSE202800 Experimental models: Organisms/strains Mice: C57BL/6J Jackson Labs Cat#000664 Mice: C57BL/6J; CD36ko Jackson Labs Cat#019006 Oligonucleotides See Table S8 This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/download.html Fiji software plug-in Adiposoft Galarraga et al. (2012) https://imagej.net/plugins/adiposoft GraphPad Prism 9 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Custom RNAseq analysis codes This paper https://doi.org/10.5281/zenodo.6588146 Open in a separate window KEY RESOURCES TABLE All original code has been deposited on github and is publically available as of the data of publication.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Quantitation Assay, SYBR Green Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: PGD2 receptors are expressed in human primary nasal epithelial cells. Cellular localization of PGD2 receptors (DP1 and CRTH2) and MUC5AC proteins in human nasal epithelial cells was analyzed using double-immunocytofluorescence staining with antibodies against MUC5AC and PGD2 receptors (DP1 and CRTH2). Immunostaining of MUC5AC (red) and DP1 (green) is shown. Merged images show co-localization of MUC5AC and DP1 in yellow (middle panels). Immunostaining of MUC5AC (red) and CRTH2 (green) is shown. Overlays of the images show co-localization of MUC5AC and CRTH2 in yellow (lower panels). No staining was detected when both primary antibodies were omitted (upper panels) (Bars indicate 10 μm).

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Staining, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: PGD2 induces MUC5B expression in NHNE and human lung mucoepidermoid carcinoma (NCI-H292) cells. A, NHNE cells were pretreated apically and basolaterally with PGD2 at the indicated concentrations for 24 h. Cell lysates were harvested for real-time quantitative RT-PCR. B, NCI-H292 cells were treated with PGD2 (0.01, 0.1, and 1 μm) for 24 h. Cell lysates were harvested for real-time quantitative RT-PCR. C, cells were treated with PGD2 (1 μm) for 1, 3, 6, 12, and 24 h, and total RNA were isolated. MUC5B expression relative to that of β2M was determined using real-time quantitative RT-PCR. D, we measured MUC5B levels in NHNE using a MUC5B ELISA kit. MUC5B levels were standardized according to the concentration of total protein for each sample. NHNE cells were pretreated apically and basolaterally with PGD2 at the indicated concentrations for 24 h. E, NCI-H292 cells were treated with PGD2 (0.01, 0.1, and 1 μm) for 24 h. Cell lysates were harvested for ELISA. F, NCI-H292 cells were treated with PGD2 (1 μm) for 1, 3, 6, 12, and 24 h. The mean values of MUC5B estimated by ELISA in cell lysates and its subgroups are expressed as ng of standard reactivity/ml of diluted resolubilized mucin sample. Significance was determined by the Mann-Whitney U test. Data are expressed as the mean ± S.D. of triplicate cultures. At least three separate experiments were performed for each measurement. C, untreated control; *, p < 0.05 versus untreated control.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY, Control

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: DP1 is required for PGD2-induced MUC5B gene expression in NCI-H292 cells. A, cells were treated with S5751 (DP1 antagonist, final concentration 0.01–10 μm) for 1 h before stimulation with 1 μm PGD2. B, cells were treated with OC0459 (CRTH2 antagonist, final concentration 0.01–10 μm) for 1 h before stimulation with 1 μm PGD2. Cells were subsequently stimulated with 1 μm PGD2 for 24 h. MUC5B expression relative to that of β2M was determined using real-time quantitative RT-PCR. Values represent the mean ± S.D. The results represent three independent experiments. C, untreated control; *, p < 0.05 versus untreated control; **, p < 0.001 versus PGD2 treatment alone.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Gene Expression, Concentration Assay, Expressing, Quantitative RT-PCR, Control

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: PGD2 induces MUC5B gene expression via ERK MAPK signaling. A, confluent cells were treated with PGD2 (1 μm) for 5, 10, 20, 30, and 60 min, and cell lysates were harvested for Western blot analysis. B, quantitation of the p-ERK/ERK ratio is shown. Representative Western blots using phospho-specific antibodies show transient activation of ERK1/2, with a maximum effect at 5 min. ERK1/2, total ERK1/2. C, cells were pretreated for 1 h with PD98059 (30 μm) and stimulated for 5 min with PGD2 (1 μm) before collection of cell lysates for Western blot analysis. D, confluent cells were pretreated for 1 h with PD98059 (30 μm) and stimulated for 24 h with PGD2 (1 μm) before collection of total RNA for real-time quantitative RT-PCR. E, after transfection with siERK1 (40 nm), siERK2 (40 nm), or siRNA-negative control (40 nm), cells were stimulated with PGD2 (1 μm) for 5 min followed by analysis of proteins using Western blotting. F, transfected cells were stimulated with PGD2 (1 μm) for 24 h before the collection of total RNA for real-time quantitative RT-PCR. MUC5B expression relative to that of β2M was determined using real-time quantitative PCR. The figures are representative of at least three independent experiments. Data are expressed as the mean ± S.D. C, untreated control; *, p < 0.05 versus PGD2 treatment alone.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Gene Expression, Western Blot, Quantitation Assay, Activation Assay, Quantitative RT-PCR, Transfection, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: DP1 is essential for PGD2-induced MUC5B expression through ERK signaling in NCI-H292 cells. A, cells were transiently transfected with constructs expressing siRNA for DP1, CRTH2, DP1 + DP2, or control siRNA. Cells were serum-starved and treated with 1 μm PGD2 for 5 min, after which cell lysates were harvested for Western blot analysis with anti-ERK1/2. B, cells were serum-starved and treated with 1 μm PGD2 for 24 h, after which cell lysates were harvested for real-time quantitative RT-PCR. MUC5B expression was determined relative to that of β2M using real-time quantitative PCR. C, untreated control; *, p < 0.05 versus PGD2 treatment alone.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Expressing, Transfection, Construct, Control, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: RSK1 is essential for PGD2-induced MUC5B gene expression. A, confluent and quiescent cells were stimulated with PGD2 (1 μm) in a time-dependent manner for up to 1 h followed by harvesting of cell lysates and Western blot analysis using anti-phospho-RSK1. β-Actin served as an internal control. B, quantitation of the p-ERK/ERK ratio is shown. The results of Western blot analyses are representative of three separate experiments. C, cells were transiently transfected with RSK1 siRNA constructs. Transfected cells were serum-starved overnight before treatment with PGD2 (1 μm) for 30 min, after which cell lysates were harvested for Western blot analysis using specific antibodies. D, 24 h after transfection, cells were treated with PGD2 (1 μm) and harvested for real time quantitative RT-PCR. MUC5B expression was determined relative to β2M by real-time quantitative PCR. Figures represent three independent experiments. Data are expressed as mean ± S.D. C, untreated control; *, p < 0.05 versus PGD2 treatment alone.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Gene Expression, Western Blot, Control, Quantitation Assay, Transfection, Construct, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: CREB mediates PGD2-induced MUC5B gene expression independent of Ca2+ signaling. A, confluent and quiescent cells were treated with PGD2 (1 μm) for 5, 10, 20, 30, and 60 min, and cell lysates were processed for Western blot analysis using phospho-specific antibodies. β-Actin was used as an internal control. B, quantitation of the p-CREB/CREB ratio is shown. The results of Western blot analyses are representative of three separate experiments. C, cells were transfected with siCREB (40 nm) or siRNA-negative control (40 nm) and stimulated with PGD2 (1 μm) for 10 min before Western blot analysis. D, cells were serum-starved overnight, stimulated with PGD2 (1 μm) for 24 h, and analyzed using real time quantitative RT-PCR. MUC5B expression was determined relative to that of β2M using real-time quantitative PCR. The figures are representative of three independent experiments. Data are expressed as the mean ± S.D. C, untreated control; *, p < 0.05 versus PGD2 treatment alone. E, fluorescence images are shown to indicate interaction between two proteins. Staining with primary anti-CREB goat polyclonal and anti-RSK1 rabbit polyclonal antibodies was followed by species-specific secondary antibodies with PLA probes. PLA signaling (red dots) was detected in NCI-H292 cells using confocal microscopy (60×). F, cells were serum-starved overnight and then treated with the indicated concentrations of PGD2 for 1 h, after which cAMP production was measured. The values shown are the means ± S.D. of experiments performed in triplicate. G, shown are fluorescence changes in response to PGD2 in NCI-H292 cells (10 μm). PGD2 in DMSO was added to Fluo-2/AM-labeled cells (arrow 1), and ATP (100 μm) was added after 8 min (arrow 2). The mean basal level of [Ca2+]i was measured for 1 min before any treatment. Blue areas represent basal [Ca2+]i, and green and red areas represent an increase from base line. [Ca2+]i versus time is shown for 10 cells (left). Colored lines show the trace of [Ca2+]i mobilization in individual cells (right). Results are representative of two independent experiments.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Gene Expression, Western Blot, Control, Quantitation Assay, Transfection, Negative Control, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Staining, Confocal Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d -Prostanoid Receptor Signaling *

doi: 10.1074/jbc.M111.247684

Figure Lengend Snippet: PGD2 enhances MUC5B promoter activity via cis-acting CRE regulatory motifs. A, cells were transiently transfected with various MUC5B promoter luciferase reporter constructs and stimulated with PGD2 (1 μm) for 24 h. Luciferase activity was measured in PGD2-treated and untreated cells. *, p < 0.05 versus −530/+92 reporter construct. B, cells were transfected with MUC5B promoter constructs containing mutated CRE sites as indicated. Cells were treated with PGD2 (1 μm) for 24 h and then harvested. The luciferase activities shown have been corrected for transfection efficiency using the β-galactosidase activity of the cell lysates. Data are expressed as the mean ± S.D. of experiments performed in triplicate cultures. *, p < 0.05 versus wild-type treated with PGD2 (1 μm). C, cells were stimulated with PGD2 (1 μm) for 20 min. Cross-linked protein-DNA complexes were immunoprecipitated using CREB antibody or normal rabbit IgG that either amplified the CREB binding flanking region in MUC5B promoter (CRE site) or a region farther upstream that did not contain a CREB-binding site (Non-CRE site). The immunoprecipitated chromatin was analyzed with PCR using primers specific to the indicated site. Input chromatin represents a portion of the sonicated chromatin before immunoprecipitation. Control IgG (C), goat anti-rabbit IgG (negative control for ChIP). D, the results were analyzed using real-time PCR and are shown as the percentage of input. The results are shown as means ± S.D. calculated from three independent experiments.

Article Snippet: Concentrations of MUC5B levels in the samples were quantified using a MUC5B enzyme immunoassay kit according to the manufacturer's directions (Uscn Life Science Inc.).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Immunoprecipitation, Amplification, Binding Assay, Sonication, Control, Negative Control, Real-time Polymerase Chain Reaction