Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: δ-opioid receptor (DOR)-expressing HEK293 cells and HEK293 cells stained with anti-DOR antibody and followed with FITC-conjugated anti-mouse IgG ( A ). Volcano plot of identified differentially expressed genes (DEGs) in DOR-HEK293 cells after 1 h treatment with CM-10 ( B ). Red boxes show changed gene expression over 1.4-time higher and less than 0.71 with p -value predicted by EdgeR of less than 0.05.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Expressing, Staining, Gene Expression

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: Predicted DOR agonistic signaling networks in DOR-HEK293 cells after 1 h treatment with CM-10. Fourteen genes with suggested involvement in cAMP-dependent protein kinases, transcriptional regulators in response to cAMP, circadian rhythm, stress and depression are shown in and were applied for network analysis by STRING. Red: circadian rhythm, Green: regulation of transcription of Notch receptor target, Yellow: PKA activation in glucagon signalling.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Activation Assay

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblot analysis of KDM4C, c-Myc, and its target genes in U87 cells transfected with c-Myc (0.6 μg) without or with the treatment of SD70 (10, 20 μM). B Immunoblot analysis of KDM4C, p53, and its target genes in shCtrl NT and shp53 expressing U87 cells without or with the treatment of SD70 (5 μM).

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B The activity of p21 and Bax luciferase reporter constructs in U87 cells treated with 0.1% DMSO (control) and SD70 following transfection with p53. *** p < 0.001 vs. pcDNA control; $$$ p < 0.001 vs. pcDNA-p53 group, ### p < 0.001 vs. DMSO + pcDNA-p53 group; one-way ANOVA. C The activity of a p21 luciferase reporter construct in U87 cells following transfection with p53 and KDM4C. D – G RT-qPCR showing KDM4s, p53, and p53 target gene mRNA levels in U87 cells treated with shRNA ( D and E ) or SD70 ( F and G ). mRNA expression levels were normalized to GAPDH mRNA levels. H Immunoblotting of KDM4C, p53, and p21 expression in U87 and U251 cells treated with shRNA or SD70 for 24 h. I ChIP-qPCR analysis of p53 and p53K372me1 levels at the PUMA promoter in 0.1% DMSO (control) and SD70 (5 µM) treated U87 cells. Data present the mean ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. shCtrl NT or DMSO control; Student’s t- tests.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Activity Assay, Luciferase, Construct, Control, Transfection, Quantitative RT-PCR, shRNA, Expressing, Western Blot, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B Immunoblotting of KDM4C, p53, and p53K372me1 in U87 and U251 cells transfected with ( A ) shRNA and ( B ) siRNA. C , D Lysates of cells treated with SD70 (20 μM) ( C ) or HA-KDM4C ( D ) were immunoprecipitated with p53 antibody followed by immunoblotting with antibodies as indicated. Ten percent of total cell lysates used in immunoprecipitation is shown as input. The level of p53K372me1 was quantified relative to p53, and the control levels were set at 1. The asterisk indicates the expected sizes for p53K372me1.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, shRNA, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblotting of KDM4C, c-Myc, and p53 in U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. B Liquid colony formation assays of U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. * p < 0.05 and ** p < 0.01 vs. Dox (-) group; one-way ANOVA. C – E Xenograft assays of U87 cells with inducible expression of shCtrl NT or shKDM4C ( n = 3). C Representative U87 xenograft tumors isolated from individual animals. D Increase in tumor volume over time. E Final tumor weights of the U87 xenografts. Data present the mean ± S.D. * p < 0.05 and ** p < 0.01 vs. shCtrl NT control; one-way ANOVA. F Representative image of IHC staining of KDM4C and c-Myc in tissues from U87 xenograft model. Scale bar, 100 μm. G Molecular model describing that KDM4C can regulate glioblastoma proliferation and apoptosis by modulating c-Myc and p53.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, Isolation, Control, Immunohistochemistry

Journal: Journal of Clinical Medicine

Article Title: Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants

doi: 10.3390/jcm11206218

Figure Lengend Snippet: XCELLigence analysis of real-time cell proliferation of untreated MG63 ( A ), untreated hMSC ( B ) and after the administration of modafinil (11.2 μg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 ng/mL) to hMSC and MG63 for 100 h. Shown are means of normalized cell index ± SD of three independent experiments with six biological replicates.

Article Snippet: The human osteoblast MG63 cell line was obtained from CLS (Cell Line Service, Eppelheim, Germany, product, no. 300441) and was maintained in DMEM-F12 (Gibco, Paisley, UK) with the additions of 10%FCS and 1 U/mL Pen/Strep. (Gibco, Paisley, UK).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants

doi: 10.3390/jcm11206218

Figure Lengend Snippet: Effect of starting cell number after administration of untreated modafinil (11.2 μg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 ng/mL) on spheroid size and morphology of MG63 (5000 cells) ( A , B ) and MG63 (8000 cells) ( C , D ). Spheroids were cultured for 21 days. Phase- contrast images of spheroids were taken at day 7, 14 and 21. Shown are means of spheroid size ± SD of three independent experiments with six biological replicates. Densitometric quantification of spheroid size was calculated by ImageJ software. Significance in difference between the groups was determined by ANOVA followed by Tukey’s post hoc test. Scale bar 250 µm.

Article Snippet: The human osteoblast MG63 cell line was obtained from CLS (Cell Line Service, Eppelheim, Germany, product, no. 300441) and was maintained in DMEM-F12 (Gibco, Paisley, UK) with the additions of 10%FCS and 1 U/mL Pen/Strep. (Gibco, Paisley, UK).

Techniques: Cell Culture, Software

Journal: Journal of Clinical Medicine

Article Title: Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants

doi: 10.3390/jcm11206218

Figure Lengend Snippet: Osteogenic differentiation of mesenchymal stem cell spheroids for 28 days. Alizarin red stained images of untreated, modafinil (11.2 μg/mL)-, atomoxetine (0.9 µg/mL)- and guanfacine- (17.7 ng/mL) treated osteogenic spheroids. Three independent experiments with six biological replicates were carried out. Alizarin red (A) stained images of untreated, modafinil- (11.2 μg/mL), atomoxetine- (0.9 µg/mL) and guanfacine (17.7 ng/mL)- treated MG63 spheroids. Three independent experiments with six biological replicates were carried out. Scale bar 250 µm.

Article Snippet: The human osteoblast MG63 cell line was obtained from CLS (Cell Line Service, Eppelheim, Germany, product, no. 300441) and was maintained in DMEM-F12 (Gibco, Paisley, UK) with the additions of 10%FCS and 1 U/mL Pen/Strep. (Gibco, Paisley, UK).

Techniques: Staining

Journal: Journal of Clinical Medicine

Article Title: Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants

doi: 10.3390/jcm11206218

Figure Lengend Snippet: Alizarin red stained images of untreated, modafinil (11.2 μg/mL)-, atomoxetine- (0.9 µg/mL) and guanfacine- (17.7 ng/mL) treated MG63 spheroids. Three independent experiments with six biological replicates were carried out. Scale bar 250 µm.

Article Snippet: The human osteoblast MG63 cell line was obtained from CLS (Cell Line Service, Eppelheim, Germany, product, no. 300441) and was maintained in DMEM-F12 (Gibco, Paisley, UK) with the additions of 10%FCS and 1 U/mL Pen/Strep. (Gibco, Paisley, UK).

Techniques: Staining

Journal: Journal of Clinical Medicine

Article Title: Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants

doi: 10.3390/jcm11206218

Figure Lengend Snippet: Cell viability assay of MG63-monolayer, using a live/dead staining with FDA. (green, live cells) and PI (red, dead cells). Images represent 7, 14 and 21-day-old monolayer MG63 of untreated, modafinil- (11.2 μg/mL), atomoxetine- (0.9 µg/mL) and guanfacine- (17.7 ng/mL) treated cells. Shown are three independent experiments with six biological replicates. Densitometric quantification of viable (green) and nonviable (red) cells was calculated by ImageJ software. Significance in difference between the groups was determined by ANOVA followed by Tukey’s post hoc test. Scale bar 150 µm.

Article Snippet: The human osteoblast MG63 cell line was obtained from CLS (Cell Line Service, Eppelheim, Germany, product, no. 300441) and was maintained in DMEM-F12 (Gibco, Paisley, UK) with the additions of 10%FCS and 1 U/mL Pen/Strep. (Gibco, Paisley, UK).

Techniques: Viability Assay, Staining, Software