Journal: Stem cells (Dayton, Ohio)

Article Title: Human mesenchymal stem/stromal cells (hMSCs) cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs stimulated macrophages into an anti-inflammatory phenotype

doi: 10.1002/stem.1191

Figure Lengend Snippet: (A,B) Effects of fractions of hMSC spheroid-conditioned medium on production of mTNFα (A) and mIL10 (B) by LPS-stimulated macrophages. (C) Relative gene expression levels of molecules involved in PGE2 synthesis from microarray data from 3 different preparations of hMSCs from 2 different donors. hMSCs plated at a low density (100–150 cells/cm2) and grown for 7 d were used as a baseline. (D–G) Real-time PCR expression data for PTGS2 (D), PTGES (E), PLA2G4A (F), and PLA2G4C (G) in hMSC monolayers and spheroids. hMSCs plated at a low density and grown for 7 d were used as a baseline. (H) PGE2 content of hMSC spheroid-conditioned medium fractions. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to vehicle control (CCM) in A, B, and H and compared to Adh Low in D, E, F, and G. Abbreviations: Boil, denatured hMSC Sph CM; Combo, equal combination of all the fractions; D1, donor 1; k, kDa; PBS, phosphate buffered saline. Other abbreviations as in Fig. 1.

Article Snippet: The test reagents were 1:10–1:10,000 dilutions of hMSC or hDF conditioned medium, 0.01–100 ng/ml PGE2 (Sigma), 0.1–10 μg/ml of a PGE2 neutralizing antibody (Cayman Chemical), 10–1,000 ng/ml of prostaglandin D2 (PGD2, Cayman Chemical), 0.01–10 μM of EP1 (SC-19220, Cayman Chemical), EP2 (AH 6809, Cayman Chemical), EP3 (L-798,106, Tocris Bioscience), and EP4 (L-161,982 and GW627368X, Cayman Chemical) receptor antagonists, 0.0001–10 ng/ml of EP4 receptor agonists (CAY10589 and CAY10580, Cayman Chemical), or 0.4–50 μM cAMP analog (8-Bromo cAMP, Tocris Bioscience).

Techniques: Gene Expression, Microarray, Real-time Polymerase Chain Reaction, Expressing, Control, Saline

Journal: Stem cells (Dayton, Ohio)

Article Title: Human mesenchymal stem/stromal cells (hMSCs) cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs stimulated macrophages into an anti-inflammatory phenotype

doi: 10.1002/stem.1191

Figure Lengend Snippet: (A-C) COX-2 inhibition in hMSC spheroids reduces the production of PGE2 by the hMSCs (A), and the conditioned medium fails to reduce mTNFα (B) and increase mIL10 (C) secretion by the stimulated macrophages. COX-1 and 2 inhibitor concentrations are shown in μM. (D–F) COX-2 knockdown in hMSC spheroids reduces the production of PGE2 by the hMSCs (D), and the conditioned medium fails to reduce mTNFα (E) and increase mIL10 (F) secretion by the stimulated macrophages. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control (DMSO) in A, B, and C and compared to control (Scr) in D, E, and F. Abbreviations: All, combination of COX-2 siRNA #1, 2, and 3; DMSO, dimethyl sulfoxide; inh, inhibitor; Scr, negative control siRNA; Tran, Transfection reagent control; #1, 2, and 3, three different siRNAs for COX-2. Other abbreviations as in Fig. 1.

Article Snippet: The test reagents were 1:10–1:10,000 dilutions of hMSC or hDF conditioned medium, 0.01–100 ng/ml PGE2 (Sigma), 0.1–10 μg/ml of a PGE2 neutralizing antibody (Cayman Chemical), 10–1,000 ng/ml of prostaglandin D2 (PGD2, Cayman Chemical), 0.01–10 μM of EP1 (SC-19220, Cayman Chemical), EP2 (AH 6809, Cayman Chemical), EP3 (L-798,106, Tocris Bioscience), and EP4 (L-161,982 and GW627368X, Cayman Chemical) receptor antagonists, 0.0001–10 ng/ml of EP4 receptor agonists (CAY10589 and CAY10580, Cayman Chemical), or 0.4–50 μM cAMP analog (8-Bromo cAMP, Tocris Bioscience).

Techniques: Inhibition, Knockdown, Control, Negative Control, Transfection

Journal: Stem cells (Dayton, Ohio)

Article Title: Human mesenchymal stem/stromal cells (hMSCs) cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs stimulated macrophages into an anti-inflammatory phenotype

doi: 10.1002/stem.1191

Figure Lengend Snippet: (A,B) PGE2 reduces the secretion of mTNFα (A) and increases the secretion of mIL10 (B) by LPS-stimulated macrophages in a dose-dependent manner. PGE2 doses are shown in ng/ml. (C,D) PGE2 neutralization in hMSC spheroid-conditioned medium reduces the anti-inflammatory effect of the conditioned medium on LPS-stimulated macrophages measured as mTNFα (C) and mIL10 (D) secretion. PGE2 antibody doses are shown in μg/ml. (E,F) PGD2 does not reduce the secretion of mTNFα (E) or increase the secretion of mIL10 (F) by LPS-stimulated macrophages. PGE2 and PGD2 doses are shown in ng/ml. Values are mean ± SD (n = 3). ns P ≥ 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control (EtOH) in A, B, E, and F and compared to control (IgG) in C and D. Abbreviations: Ab, antibody; EtOH, ethanol; PBS, phosphate buffered saline. Other abbreviations as in Fig. 1.

Article Snippet: The test reagents were 1:10–1:10,000 dilutions of hMSC or hDF conditioned medium, 0.01–100 ng/ml PGE2 (Sigma), 0.1–10 μg/ml of a PGE2 neutralizing antibody (Cayman Chemical), 10–1,000 ng/ml of prostaglandin D2 (PGD2, Cayman Chemical), 0.01–10 μM of EP1 (SC-19220, Cayman Chemical), EP2 (AH 6809, Cayman Chemical), EP3 (L-798,106, Tocris Bioscience), and EP4 (L-161,982 and GW627368X, Cayman Chemical) receptor antagonists, 0.0001–10 ng/ml of EP4 receptor agonists (CAY10589 and CAY10580, Cayman Chemical), or 0.4–50 μM cAMP analog (8-Bromo cAMP, Tocris Bioscience).

Techniques: Neutralization, Control, Saline

Journal: Stem cells (Dayton, Ohio)

Article Title: Human mesenchymal stem/stromal cells (hMSCs) cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs stimulated macrophages into an anti-inflammatory phenotype

doi: 10.1002/stem.1191

Figure Lengend Snippet: (A) Hierarchical clustering of hMSC and hDF microarray data. (B) Relative gene expression levels of molecules involved in PGE2 synthesis from the hMSC and hDF microarray data. hMSCs plated at a low density and grown for 7 d were used as a baseline. (C,D) hDF spheroids do not express high levels of PTGS2 (C) or secrete PGE2 (D). (E,F) Conditioned medium from hDF spheroids does not lower the secretion of mTNFα (E) or increase the secretion of mIL10 (F) by LPS-stimulated macrophages. Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to Adh Low in C and compared to vehicle control (CCM) in D, E, and F. Abbreviation: hDF, human adult dermal fibroblast. Other abbreviations as in Fig. 1.

Article Snippet: The test reagents were 1:10–1:10,000 dilutions of hMSC or hDF conditioned medium, 0.01–100 ng/ml PGE2 (Sigma), 0.1–10 μg/ml of a PGE2 neutralizing antibody (Cayman Chemical), 10–1,000 ng/ml of prostaglandin D2 (PGD2, Cayman Chemical), 0.01–10 μM of EP1 (SC-19220, Cayman Chemical), EP2 (AH 6809, Cayman Chemical), EP3 (L-798,106, Tocris Bioscience), and EP4 (L-161,982 and GW627368X, Cayman Chemical) receptor antagonists, 0.0001–10 ng/ml of EP4 receptor agonists (CAY10589 and CAY10580, Cayman Chemical), or 0.4–50 μM cAMP analog (8-Bromo cAMP, Tocris Bioscience).

Techniques: Microarray, Gene Expression, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Human mesenchymal stem/stromal cells (hMSCs) cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs stimulated macrophages into an anti-inflammatory phenotype

doi: 10.1002/stem.1191

Figure Lengend Snippet: (1) hMSCs aggregate in a hanging drop to form a spheroid. (2) Aggregation and the resulting stress result in activation of caspases and NFκB. (3) The expression of PLA2G4A/C, PTGS2, and PTGES genes is up-regulated. (4) Phospholipases (PLA2) release arachidonic acid from plasma membrane phospholipids (PL). (5) Arachidonic acid is converted into PGH2 by COX-2. (6) PGH2 is converted into PGE2 by PGE synthase (PGES) followed by secretion of PGE2 (7) LPS stimulation of macrophages increases the secretion of pro-inflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. (8) hMSC spheroid produced PGE2 binds to the EP4 receptor on LPS-stimulated macrophages. (9) PGE2 binding results in decreased secretion of TNFα, CXCL2, IL6, IL12p40, and IL23 and increased secretion of anti-inflammatory cytokines IL10 and IL1ra, and increased surface expression of M2 macrophage marker CD206 by the stimulated macrophages. Abbreviations as in Fig. 1.

Article Snippet: The test reagents were 1:10–1:10,000 dilutions of hMSC or hDF conditioned medium, 0.01–100 ng/ml PGE2 (Sigma), 0.1–10 μg/ml of a PGE2 neutralizing antibody (Cayman Chemical), 10–1,000 ng/ml of prostaglandin D2 (PGD2, Cayman Chemical), 0.01–10 μM of EP1 (SC-19220, Cayman Chemical), EP2 (AH 6809, Cayman Chemical), EP3 (L-798,106, Tocris Bioscience), and EP4 (L-161,982 and GW627368X, Cayman Chemical) receptor antagonists, 0.0001–10 ng/ml of EP4 receptor agonists (CAY10589 and CAY10580, Cayman Chemical), or 0.4–50 μM cAMP analog (8-Bromo cAMP, Tocris Bioscience).

Techniques: Activation Assay, Expressing, Clinical Proteomics, Membrane, Produced, Binding Assay, Marker

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: In silico analysis of rod-specific photoreceptor cGMP phosphodiesterase (PDE6D) expression in hepatocellular carcinoma (HCC). ( A ) Oncomine TM human cancer microarray database analysis of six patient datasets depicting PDE6D mRNA expression levels in HCCs and non-tumorous livers (* p < 0.05 vs. non-tumorous livers). ( B , C ) Oncomine TM human cancer microarray database analysis of PDE6D expression as detected in large-scale RNA profiling studies comparing diverse carcinomas of different origins (* p < 0.05 vs. average expression).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: In Silico, Expressing, Microarray

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: PDE6D expression in HCC in vivo and in vitro. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis of HCC patient samples and paired non-tumorous liver tissues (* p < 0.05). ( B ) Detection of PDE6D mRNA in human HCC cells (PLC, Hep3B, HepG2) after qRT-PCR amplification using gel electrophoresis (left panel) and relative PDE6D mRNA levels (qRT-PCR) in human HCC cell lines (PLC, Hep3B, HepG2) compared with primary human hepatocytes derived from different donors (#1–3) (right panel) (* p < 0.05 vs. hepatocytes). ( C ) Exemplary Western blot image (left panel) and summarized densitometric quantification (right panel) of PDE6D protein levels in HCC cells (PLC, Hep3B, HepG2, Huh-7) compared with hepatocytes derived from different donors (#1–2) (* p < 0.05 vs. hepatocytes).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Quantitative RT-PCR, Amplification, Nucleic Acid Electrophoresis, Derivative Assay, Western Blot

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Effects of PDE6D knockdown on HCC proliferation and clonogenicity. Prior to functional experiments, HCC cell lines (PLC, Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis (* p < 0.05 vs. control). ( B ) PDE6D protein levels as quantified by Western blot analysis. The left panel depicts an exemplary Western blot image, and the right panel depicts the summarized densitometric quantification (* p < 0.05 vs. control). ( C ) Real-time cell proliferation (xCELLigence). Exemplary proliferation curves (left panel) and quantified “slopes” (summarizing the proliferative ability) (right panel) are shown (* p < 0.05 vs. control). ( D ) Relative (to mean) PDE6D as correlated to CyclinD1 mRNA expression levels (qRT-PCR) in human HCC patient tissue samples. ( E , F ) Anchorage-dependent clonogenic assay (an exemplary image (Hep3B) is depicted in the left panel of ( E )). Quantification of colony number (right panel of ( E )) and sizes ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Functional Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Clonogenic Assay

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and function of PDE6D in sorafenib resistance. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B and HepG2 cell clones (* p < 0.05). ( B ) Exemplary image (left panel) and summarized densitometric quantification (right panel) of Western blot analysis of PDE6D levels in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B cells (rel DM: relative (PDE6D/Actin) densitometry) (* p < 0.05 vs. non-resist.). ( C , D ) Prior to functional experiments, resistant cells (Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( C ) Depicts relative proliferation (cell numbers) and ( D ) depicts exemplary clonogenic assays. ( E , F ) Forced overexpression of PDE6D protein (PDE6D-OE) in HCC cells (e.g., PLC) was performed by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (an empty control vector without the PDE6D ORF was used as control treatment). ( E ) Depicts Western blot analysis depicting the overexpressed Myc-DDK-tagged PDE6D protein after PDE6D-OE as well as endogenous (arrow) PDE6D in both PDE6D-OE and control-treated cells. ( F ) Depicts exemplary images (representing 8 replicate values of 2 independent experiments) of cells cultured in 6-wells for 72 h (100,000 cells were initially seeded per 6-well) and treated with different doses of sorafenib (0, 4, 8 µM).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Western Blot, Functional Assay, Transfection, Over Expression, Plasmid Preparation, Cell Culture

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: TGF-â-mediated regulation of PDE6D and the effect of PDE6D on HCC cell migration. ( A – C ) Quantitative RT-PCR analysis of VIMENTIN, SNAIL, S100A4 ( A ) and KRAS ( B ) mRNA levels in HCC cells (PLC) that were stimulated with different doses of recombinant human TGF-â1 protein for 72–96 hours (* p < 0.05 vs. control). ( C , D ) Quantitative RT-PCR revealing mRNA levels ( C ) as well as protein levels as quantified by Western blot analysis (including a representative Western blot image) (the densitometric values represent two independent Western blot analysis) ( D ) of PDE6D expression in HCC cells (PLC) that were treated with different doses of recombinant human TGF-â1 for 72–96 hours. ( D ) also depicts co-treatment with 15 µM of the TGF-â-receptor-1 (TGFBR1) inhibitor LY2157299 (“galunisertib”) (* p < 0.05 vs. control). ( E , F ) Prior to Boyden chamber experiments, non-resistant HCC cells (PLC, Hep3B) ( E ) and sorafenib-resistant Hep3B cells ( F ) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). Migration (migrating cells per visual field) as measured by Boyden chamber migration assay (duration of migration: 4 hours) is depicted as absolute cell counts ( E ) or as normalized migration ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Migration, Quantitative RT-PCR, Recombinant, Western Blot, Expressing, Transfection

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and cellular localization of PDE6D in HCC in vivo and in vitro. ( A ) PDE6D staining (exemplary images) of non-tumorous liver tissues (left side) and HCC tissues (right side) deposited on the Human Protein Atlas database. ( B ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues ( N = 117) as compared with corresponding non-tumorous liver tissues ( N = 127) (Fisher’s exact P < 0.001). ( C , D ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues correlated with tumor grading (Fisher’s exact P = 0.030) ( C ) and tumor stages ( D ). ( E ) Exemplary immunohistological images of PDE6D protein expression in human HCC samples and corresponding non-tumorous liver tissues applying a tissue microarray revealing nuclear staining next to cytoplasmatic staining patterns (paired samples of two different patients (#1, #2) are depicted). ( F , G ) Tissue microarray analysis comparing tumor stages ( F ) and ERK activation (p-ERK) ( G ) in human HCC tissues with (“yes”) and without (“no”) cytoplasmatic localization pattern of PDE6D. ( H ) In silico-based analysis of importin-á-dependent nuclear localization signals (NLS, red letters ) using the “cNLS Mapper” predicted bipartite NLS in both isoforms of PDE6D (score for both isoforms was 5.3). A legend depicts that higher scores indicate stronger NLS activity and defines major localizations in dependence of each score. ( I , J ) Exemplary immunofluorescence ( I ) and Western blot analysis ( J ) depicting nuclear localization of PDE6D (I) and expression of PDE6D in both nuclear and cytoplasmatic fractions ( J ) of HCC cell lysates.

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Staining, Microarray, Activation Assay, In Silico, Activity Assay, Immunofluorescence, Western Blot

Journal: AIDS

Article Title: Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipodystrophy: implications for the pathogenesis of the disease

doi: 10.1097/qad.0b013e32833779a3

Figure Lengend Snippet: Fig. 1. Validation of the data obtained by microarray analysis on adipose tissue from lipodystrophy patients. (a) Differences in transcription levels of the indicated genes between adipose tissue from lipodystrophy patients and controls, as evaluated by real- time PCR. (b) Representative western blot of LON protease expression in adipose tissue from lipodystrophy patients and healthy controls. (c) Quantitative data, based upon densitometric analysis of all samples, are reported in the histogram, setting arbitrarily the first negative control as 1. P < 0.05, P < 0.01. C, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LD, lipodystrophy patients.

Article Snippet: The membrane was incubated in the presence of a human LON protease rabbit antisera (a kind gift from Dr K. Suzuki, University of New Jersey, New Jersey, USA), washed and incubated with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP; Serotec, Kidlington, UK).

Techniques: Biomarker Discovery, Microarray, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Negative Control, Control

Journal: Biomedicines

Article Title: Comprehensive Bioinformatics Analysis Reveals the Potential Role of the hsa_circ_0001081/miR-26b-5p Axis in Regulating COL15A1 and TRIB3 within Hypoxia-Induced miRNA/mRNA Networks in Glioblastoma Cells

doi: 10.3390/biomedicines12102236

Figure Lengend Snippet: General workflow of the bioinformatics analysis. By using limma and deseq2, we conducted differential expression (DE) analyses of three publicly available datasets deposited in the Gene Expression Omnibus (GEO). Both mRNA and miRNA data consisted of U-87 MG cells treated with hypoxia and normoxia, while circRNA data consisted of GBM patient-derived cells and neural progenitor cells as a control group. By intersecting the results of DE analyses with corresponding interaction pairs from miRTarBase and circBank, we constructed putative ceRNA networks. Note that we concurrently constructed two types of networks according to the ceRNA hypothesis (this is omitted in to keep it more concise); this consisted of upDECs/down-hiDEMs/up-hiDEGs and a second one that consisted of down-hiDECs/up-hiDEMs/downDEGs. For all significantly expressed hypoxia-induced genes (hiDEGs; both up- and downregulated), we performed GO enrichment analysis and constructed a PPI network in Cytoscape; the PPI network was further subjected to MCL clustering, and selected clusters were subsequently analyzed with GO enrichment terms. Next, by analyzing different PPI subclusters in the context of the obtained ceRNA and GO enrichment results, we selected the hsa_circ_0001081/miR-26b-5p/ TRIB3 / COL15A1 axis and its downstream targets for further investigation. To do so, we analyzed the protein-coding components of this axis by molecular subtype and survival analyses using data from Giloma-BioDP and the GDC Data Portal, respectively. For more detailed information about each step of the workflow, see the Materials and Methods section. For utilized R code and generated results, see DOI 10.5281/zenodo.13770443. Figure numbers corresponding to particular steps of the workflow are in brackets. GBM—glioblastoma; hiDEGs—hypoxia-induced genes; hiDEMs—hypoxia-induced miRNA; DECs—hypoxia-induced circRNA; up—upregulated; down—downregulated; PPI—protein–protein interaction; GO—gene ontology; ceRNA—competing endogenous RNA; GDC—Genomic Data Commons; TCGA—The Cancer Genome Atlas; Glioma-BioDP—Glioma Bio Discovery Portal; MCL—Markov Cluster Algorithm (a cluster algorithm for graphs).

Article Snippet: We extracted raw data from the following records: GSE245800, which contains microarray data from the Illumina HumanHT-12 V4.0 expression beadchip (deposited as a part of paper by de Oliveira et al. [ ]); GSE142719, which contains miRNA-seq data from the Illumina HiSeq 2500 (deposited as a part of a paper by Wang et al. [ ]) and GSE146463, which contains microarray data from the Arraystar Human CircRNA microarray V2 (deposited as a part of a paper by Bronisz et al. [ ]).

Techniques: Expressing, Derivative Assay, Control, Construct, Generated

Journal: Biomedicines

Article Title: Comprehensive Bioinformatics Analysis Reveals the Potential Role of the hsa_circ_0001081/miR-26b-5p Axis in Regulating COL15A1 and TRIB3 within Hypoxia-Induced miRNA/mRNA Networks in Glioblastoma Cells

doi: 10.3390/biomedicines12102236

Figure Lengend Snippet: Differential expression (DE) analysis of circRNA in patient-derived GBM cells vs. neural progenitor cells (NPC) and putative GBM-specific ceRNA network construction; ( A ) volcano plot for differentially expressed circRNAs (DECs) in GBM patient-derived cells as compared to neural progenitor cells (NPC), the threshold for significant circRNA (in blue): |log 2 FC| > 2 and p.adjusted < 0.05; ( B ) Sankey diagram of putative GBM ceRNA network of 3 upregulated differentially expressed circRNAs (upDECs), hypoxia-specific downregulated miR-365a-5p, and hypoxia-specific upregulated SGCD ; ( C ) Sankey diagram of putative GBM ceRNA network of downregulated differentially expressed circRNAs (downDECs), hypoxia-specific upregulated miRNAs (up-hiDEMs), and hypoxia-specific downregulated genes (down-hiDEGs).

Article Snippet: We extracted raw data from the following records: GSE245800, which contains microarray data from the Illumina HumanHT-12 V4.0 expression beadchip (deposited as a part of paper by de Oliveira et al. [ ]); GSE142719, which contains miRNA-seq data from the Illumina HiSeq 2500 (deposited as a part of a paper by Wang et al. [ ]) and GSE146463, which contains microarray data from the Arraystar Human CircRNA microarray V2 (deposited as a part of a paper by Bronisz et al. [ ]).

Techniques: Expressing, Derivative Assay

Journal: Cancer research

Article Title: Holo-retinol-binding protein and its receptor STRA6 drive oncogenic transformation

doi: 10.1158/0008-5472.CAN-14-1052

Figure Lengend Snippet: a) Levels of STRA6 mRNA in samples of normal human breast and invasive breast carcinoma (36). Data were obtained from Oncomine™ (Compedia Bioscience, Ann Arbor, Michigan). *p<0.05. b) STRA6 expression in a TissueScan™ tissue qPCR array consisting of cDNA derived from samples of normal breast and denoted stages of breast tumors (OriGene). *p<0.05. c) Relative STRA6 mRNA levels in denoted cell lines and in normal human mammary epithelial cells (HuMEC) measured by Q-PCR. #p<0.05 vs. HuMEC. d) Levels of STRA6 mRNA in samples of normal human colon and colon carcinoma (36). *p<0.05 vs. normal colon. e) Analysis of TissueScan™ tissue qPCR array consisting of cDNA derived from samples of normal colon and denoted stages of colon tumors (OriGene). *p<0.05 vs. normal colon. f) Levels of STRA6 mRNA in normal colon tissue and in colon polyps in 60 day old APCmin mice, measured by Q-PCR. Mean±S.E.M. (n=3), ‡p<0.0001 vs. normal mouse colon. g) Relative STRA6 mRNA levels in denoted human colon carcinoma cell lines and in normal human colon cells (normal), measured by Q-PCR. Data are mean±S.E.M (n=3). #p<0.05 vs. normal epithelium cells. h) Relative RBP mRNA levels in denoted cell lines and in normal human mammary epithelial cells (HuMEC) measured by Q-PCR.*p<0.05 vs. HuMEC. i) Levels of STRA6 mRNA in samples of normal human colon cells and colon carcinoma (36)). *p<0.05 vs. normal colon. j) RBP expression in a TissueScan™ tissue qPCR array consisting of cDNA derived from samples of normal colon and denoted stages of colon tumors (OriGene).*p<0.05 vs. normal colon. k) Relative RBP mRNA levels in denoted colon carcinoma cell lines and in normal human colon cells (normal), measured by Q-PCR. Data are mean±S.E.M (n=3). #p<0.05 vs. normal epithelium cells. All data are mean±S.E.M. (n=3).

Article Snippet: Accordingly, analysis of a human breast cancer cDNA array (OriGene) showed that STRA6 expression is significantly higher in breast tumors as compared with normal mammary tissue ( ).

Techniques: Expressing, Derivative Assay