Journal: International Journal of Molecular Sciences

Article Title: Quinolones Modulate Ghrelin Receptor Signaling: Potential for a Novel Small Molecule Scaffold in the Treatment of Cachexia

doi: 10.3390/ijms19061605

Figure Lengend Snippet: Quinolone compounds activate GHS-R1a receptor overexpressed in Hek cells as well as the endogenous receptor expressed in hypothalamic neurons. Quinolone compounds (10 µM) and MK-0677 activate GHS-R1a receptors in transfected human embryonic kidney (Hek293a) cells ( A ) and embryonic mouse hypothalamic cell line-N38 ( B ) by using cell calcium imaging through confocal microscopy (for visualization, see Supplementary videos in ). Significant differences compared to the baseline and MK-0677 controls are presented with * and # respectively with * indicating p ≤ 0.05 and ***/### indicating p ≤ 0.001. For further statistic details, see the methods section.

Article Snippet: Embryonic mouse hypothalamus cell line N38 (mHypoE-N38) (Cederlane Laboratories, Cellutions Biosystems, Burlington, ON, Canada) was maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) at culture conditions (37 °C and 5% CO 2 in a humidified atmosphere).

Techniques: Transfection, Imaging, Confocal Microscopy

Journal: Biomedicines

Article Title: Microbiome Modulation as a Therapeutic Approach in Chronic Skin Diseases

doi: 10.3390/biomedicines9101436

Figure Lengend Snippet: Probiotic application in chronic ulcers.

Article Snippet: 12 , Argenta et al. —2016 [ ] , Female C57 BL/b mice (n = 38) , Animal study , The mice were divided into groups. Injured sites were treated with vehicle (burn wound control), probiotics ( L. plantarum ATCC 1024) only, pathogenic bacteria ( P. aeruginosa ) only, or probiotics + pathogen ( Lactobacillus and P. aeruginosa ) for five days , Lower mortality rate and inhibition of pathogenic bacteria.

Techniques: Infection, Control, Clinical Proteomics, Bacteria, Probiotics, In Vitro, Saline, Isolation

Journal: Biomedicines

Article Title: Microbiome Modulation as a Therapeutic Approach in Chronic Skin Diseases

doi: 10.3390/biomedicines9101436

Figure Lengend Snippet: Probiotic application of burns in humans and animal models.

Article Snippet: 12 , Argenta et al. —2016 [ ] , Female C57 BL/b mice (n = 38) , Animal study , The mice were divided into groups. Injured sites were treated with vehicle (burn wound control), probiotics ( L. plantarum ATCC 1024) only, pathogenic bacteria ( P. aeruginosa ) only, or probiotics + pathogen ( Lactobacillus and P. aeruginosa ) for five days , Lower mortality rate and inhibition of pathogenic bacteria.

Techniques: Control, Cream, Infection, Translocation Assay, Saline, Probiotics, Injection, Inhibition, Suspension, Bacteria, Permeability

Journal: Cellular and Molecular Life Sciences

Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

doi: 10.1007/s00018-022-04446-4

Figure Lengend Snippet: Effects of GHS-R1α activation by ghrelin on eEF2 phosphorylation. a Proposed contribution of elongation into ribosome turnover and protein production de novo. Decreased eEF2 phosphorylation is expected to increase elongation rate, ribosome availability and protein production levels. b , c Western blotting analysis of time- and concentration-dependent changes in eEF2 (Thr56) phosphorylation induced in HEK293 GHS-R1α-EGFP+ cells by ghrelin treatment; b 100 nM ghrelin for 15–60 min and c 4–100 nM ghrelin for 30 min. d Analysis of eEF2 phosphorylation in parental HEK293 cells lacking GHS-R1α. e Analysis of GHS-R1α protein expression in mouse embryonic hypothalamus cell lines E-N38 and E-N41; in E-N41, GHS-R1α protein levels are ~ 60% higher. f Ghrelin concentration-dependent decrease in p-eEF2 (Thr56) levels in E-N41 cells (30 min treatment). g Changes in the extracellular G Luc signals triggered by ghrelin treatment in E-N41 cells (200 nM, 30 min). Cells were pre-incubated for 14–16 h in serum-free DMEM supplemented with NEAA. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin treatment time: p-eEF2 (4,20) = 124.011, p < 0.001, one-way ANOVA, p < 0.001 for 15, 30, 45 and 60 min, Dunnett post hoc test vs mock treatment ( N = 6). c Effect of ghrelin concentration: p-eEF2 (4,20) = 45.25, p < 0.001, one-way ANOVA, p < 0.001 for 3 nM, 11 nM, 33 nM and 100 nM, Dunnett post hoc test vs mock treatment ( N = 6). f effect of ghrelin concentration: p-eEF2 (5,12) = 27.815, p < 0.001, one-way ANOVA, p < 0.001 for 167 nM and 500 nM ghrelin, Dunnett post hoc test vs mock treatment ( N = 3). g effect of ghrelin treatment, N = 4, independent samples T test: G Luc t (6) = – 1.234, p = 0.181

Article Snippet: HEK293 stably expressing GHS-R1α-EGFP (HEK293 GHS-R1α-EGFP+ ) [ ] and embryonic mouse hypothalamus N38 and N41 cells (Hypo E-N38 and Hypo E-N41, Cedarlane Laboratories, Burlington, Canada) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/100 μg/ml streptomycin (P/S), non-essential amino acids (NEAA) and 10 mM HEPES, pH 7.2 (complete DMEM).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Concentration Assay, Expressing, Incubation

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: N42 and N38 hypothalamic cells use promoter I.f to regulate aromatase mRNA expression. Esr1 mRNA and ESR1 protein expression levels in N42 hypothalamic cells are higher than N38 hypothalamic cells. A) Real-time RT-PCR was performed employing a probe complementary to the exon I.f-exon II junction to measure promoter I.f-driven aromatase mRNA expression. Relative units are shown as the ratio of aromatase mRNA:Gapdh mRNA; (presented as l.f-specific aromatase mRNA/Gapdh mRNA). Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Conventional RT-PCR (B) and IB (C) were performed to measure ESR1 expression in brain tissue (positive control) and N42 or N38 hypothalamic cells. Gapdh and actin were used as loading controls. The figures represent one of three independently performed experiments.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: Estradiol regulates aromatase mRNA expression and enzyme activity in N42, but not N38, hypothalamic cells. A) Real-time RT-PCR was performed to measure aromatase mRNA expression after 6, 12, and 24 h of E2 (10−7 M) treatment. Aromatase mRNA levels were normalized to Gapdh mRNA levels. Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase activity assays were performed in N42 (B) and N38 (C) hypothalamic cells after 6, 12, and 24 h of E2 (10−7 M) treatment. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase enzyme activity in each neuronal line treated with the aromatase enzyme inhibitor letrozole (LET) was undetectable (data not shown).

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: The E2 antagonist ICI inhibits baseline and E2-induced aromatase mRNA expression and enzyme activity in N42 hypothalamic cells. A) Real-time RT-PCR was performed after cells were incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. B) Aromatase activity assays were performed to measure enzyme activity in cells incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase activity in the N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown).

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Incubation

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: AP-1 cis-regulatory elements within the nt −200/−1 region of promoter I.f are essential for E2-dependent induction of aromatase expression in N42 hypothalamic cells. A) Serial deletion analysis was performed to localize the E2-responsive regions of aromatase promoter I.f. The promoter I.f-luciferase (LUC) reporter constructs were named according to the nucleotide positions of their 5′ termini. All constructs were transiently transfected into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence or absence of 10−7 M E2. Mean luciferase activity of each construct is given relative to the nt −1000/−1 promoter-luciferase reporter construct. Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, paired t-test). B) Mutational analysis of promoter I.f was performed to verify functional cis-regulatory elements. Substitution mutations were made within two putative AP-1 cis-regulatory elements in the −200/−1 promoter-luciferase reporters. Constructs were transiently cotransfected with ESR1 expression vector into N42 hypothalamic cells, and luciferase activities were measured after treatment with vehicle or 10−7 M E2. Results shown are the mean ± SEM of three independent experiments (**P < 0.01, ANOVA).

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Luciferase, Construct, Transfection, Activity Assay, Functional Assay, Plasmid Preparation

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ChIP analysis reveals ESR1 and JUN, but not FOS, bind to promoter I.f in N42 hypothalamic cells. We performed ChIP assays using N42 hypothalamic cell extracts treated with vehicle or 10−7 M E2 for 6 h. Sonicated cell supernatant was used as input DNA (positive control). Precleared chromatin was used for IP reactions with a rabbit polyclonal antibody directed against human (A) ESR1 and (B) JUN or FOS, as well as normal rabbit IgG. ESR1 was recruited to the E2-responsive nt −193/−47 but not to a more distal (nt −925/−768) region (see Fig. 4). Estradiol induced the recruitment of JUN but not FOS to the nt −193/−47 region. The figures represent one of three independently performed experiments.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Sonication, Positive Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: Putative AP-1 sites at nt −130/−124 and nt −166/−160 regions of promoter I.f are the major c-Jun-ESR1 binding sites. An EMSA was used to visualize JUN and ESR1 binding to AP-1 sites within promoter I.f. The EMSAs were performed using 5 μg of nuclear extract from N42 hypothalamic cells treated with E2 (10−7 M; 6 h) and radiolabeled oligonucleotide probes containing critical AP-1 sites and flanking sequences. A) Binding to an oligonucleotide identical to the nt −138/−114 sequence in promoter I.f and containing the nt −130/−124 AP-1 element (native; lane 2). Competition studies were performed using 100-fold excess unlabeled wild-type (consensus; lanes 3 and 4) or native oligonucleotides (lane 5) or mutant native oligonucleotides (lane 6). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 7 and 9) or 0.5 μg of anti-ESR1 (lanes 8 and 10) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 11 and 12). B) Binding to an oligonucleotide identical to the nt −175/−151 sequence in promoter I.f and containing the nt −166/−160 AP-1 element (native) (lane 2). Competition studies were performed using 100-fold excess unlabeled consensus or native probes (lanes 5 and 7) or mutant consensus or mutant native probes (lanes 6 and 8). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 10 and 12) or 0.5 μg of anti-ESR1 (lanes 9 and 11) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 13 and 14). Black arrows indicate JUN-ESR1 complexes, and white arrows indicate supershifted complexes by either c-Jun or ESR1 antibodies. The figures represent one of three independently performed experiments.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Binding Assay, Sequencing, Mutagenesis, Immunodepletion, Negative Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: The ESR1 DNA-binding domain is not required for mediating E2-dependent aromatase expression in N42 hypothalamic cells. The nt −200/−1 I.f promoter-luciferase reporter construct was transiently cotransfected with either wild-type ESR1 or ESR1 carrying a mutation at DNA-binding domain (ESR1-AA) into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence of E2 (10−7 M). Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, ANOVA).

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Binding Assay, Expressing, Luciferase, Construct, Mutagenesis

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ESR1 and JUN, but not FOS, form a complex in N42 hypothalamic cells. A) Immunoprecipitation with antibodies against human JUN or FOS and IB with an antibody against ESR1 (mESR1; raised in mouse, monoclonal) were performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). B) Immunoprecipitation with two different ESR1 antibodies, mESR1 or rESR1 (raised in rabbit, polyclonal), followed by IB with an antibody against JUN was performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). Immunoprecipitation with a nonspecific rabbit IgG was the negative control. The figures represent one of three independently performed experiments.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Immunoprecipitation, Negative Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ESR1 mediates E2-stimulated aromatase mRNA expression in N42 hypothalamic cells. N42 hypothalamic cells were transfected with Esr1 siRNA or with nonspecific siRNA as a negative control (NS) and were cultured for an additional 48 h. A) Aromatase mRNA levels were analyzed by real-time RT-PCR in the presence or absence of E2 (10−7 M; 6 h). Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3; **P < 0.01, ANOVA. B) Aromatase activity assays were performed in the presence or absence of E2 (10−7 M; 6 h). The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase enzyme activity in N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown). Real-time RT-PCR (C) and IB (D) were performed to measure Esr1 mRNA and protein levels, respectively. Actin was used as a loading control. The IB figure represents one of three independently performed experiments. Real-time RT-PCR results are expressed as the mean ± SEM (n = 3), and Gapdh was used as a loading control. **P < 0.01, ANOVA. Representative results from three independent experiments are shown.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Transfection, Negative Control, Cell Culture, Quantitative RT-PCR, Activity Assay, Control

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-CART (55-102) Phoenix Pharmaceuticals H-003-62, RRID:AB_2313614 Rabbit anti-pS6 (Ser235/236) Cell Signaling #2211, RRID:AB_331679 Rabbit anti-pSTAT3(Y705) Cell Signaling #9131, RRID:AB_331586 Mouse anti-Thrombospondin (A6.1) Santa Cruz Sc-59887, RRID:AB_793045 Goat anti-GFP Abcam Ab6673, RRID:AB_305643 Rabbit anti-GFP Millipore Ab3080, RRID:AB_91337 Chicken anti-Beta-3 tubulin Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab107216","term_id":"30250552","term_text":"AB107216"}} Ab107216 , RRID:AB_10899689 Rabbit anti-Beta-3 tubulin Sigma-Aldrich T2200, RRID:AB_262133 Rabbit anti-RFP Rockland 600-401-379, RRID:AB_2209751 Rat anti-HA Roche 11867423001, RRID:AB_390918 Rabbit anti-RBPMS PhosphoSolutions 1830-RBPMS, RRID:AB_2492225 Mouse anti-osteopontin R & D System AF808, RRID:AB_2194992 Rabbit anti-melanopsin Advanced Targeting System AB-N38, RRID:AB_1608077 Bacterial and Virus Strains AAV2-Cre Boston Childrens Hospital Viral Core NA AAV-shH10-GFAP-GFP Park Lab NA AAV-ShH10-GFAP-THBS1 Park Lab NA AAV2-THBS1 and THBS4 (various forms) Park Lab NA AAV2-RGS4, VTN, CD86 and various shRNA Park Lab NA Biological Samples Chemicals, Peptides, and Recombinant Proteins KRFYVVMWKK GenScript NA KRFYGGMWKK GenScript NA Critical Commercial Assays RNAscope Multiplex Fluorescent Reagent Kit V2 ACD-Bio 323100 SMART-Seq v4 Ultra Low Input RNA kit Clontech 634888 Deposited Data Raw and analyzed data Present manuscript GEO: {"type":"entrez-geo","attrs":{"text":"GSE115661","term_id":"115661"}} GSE115661 Experimental Models: Cell Lines Human: HEK293T cells ATCC CRL-3216, RRID:CVCL_0063 Mouse: mouse embryonic fibroblasts ATCC A gift from Dr. A. Capobianco Neuro-2a cells ATCC CCL-131 RRID:CVCL_0470 Experimental Models: Organisms/Strains Mouse: Opn4Cre Ecker et al. 2010 NA Mouse: Opn4CreERT Chen et al. 2011 NA Mouse: C57BL/6J Jackson Labs 000664, RRID:IMSR_JAX:000664 Mouse: B6.129S4- Pten tm1Hwu /J Jackson Labs 006440, RRID:IMSR_JAX:006440 Mouse: R26 loP-STOP-loxP-tdTomato Dr.

Techniques: shRNA, Recombinant, Multiplex Assay, Clone Assay, Software