Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: The 8701-BC cells express a soluble factor that is responsible of HAS2 induction in NHDF. ( A ) Quantitative RT-PCR analysis of HAS2 gene in NHDF and NHDF cultured in 8701-BC 48 h CM. The results are expressed as fold change with respect to NHDF cultured in RPMI1640 and each bar represents mean ± S.D. of two independent experiments. * p < 0.05. ( B ) SDS PAGE after Blue Coomassie staining of different amounts in term of total proteins (2.5, 5, 10 μg respectively) of 8701-BC 48 h CM. Bands at MW of approximately 55 kDa were excised and analyzed by MALDI-TOF as reported in Material and Methods. ( C ) Quantitative RT-PCR analysis of the c10orf118 (Q7z3E2) gene expression level in different breast cancer cell lines (MCF-7, MDA-MB-231 and 8701-BC), expressed as fold change with respect to NHDF. Bars represent mean ± S.D. of triplicate samples. * p < 0.05, *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Quantitative RT-PCR, Cell Culture, SDS Page, Staining, Gene Expression

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: c10orf118 silencing influences HA-related genes expression in MCF-7. Quantitative RT-PCR analyses of MCF-7 cells nucleofected for 48 h with 30 pmol of c10orf118 siRNA or a scrambled siRNA. The graphs represent the mRNA levels of ( A ) c10orf118, ( B ) HAS2, ( C ) the long non-coding RNA HAS2-AS1 and ( D ) the HA receptor CD44. Data are represented as mean ± S.D. of six independent experiments. ** p < 0.01, and *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: Effects of fibroblasts/breast cancer cells co-culture on HA production and HAS2 gene expression. ( A ) HA secretion detected by HPLC in the culture medium and ( B ) HAS2 expression in NHDF co-cultured with MCF-7 and MDA-MB-231 cells (see Materials and Methods). Bars represent mean ± S.D. of triplicate samples. ( C ) Quantitative RT-PCR of HAS2 expression in NHDF and MCF-7 cells. ( D ) Relative gene expression of HAS2 and ( E ) HAS3 in MCF-7 cells grown for 24 h in the absence (CNTR) or presence of NHDF conditioned media (+ CM NHDF). * p < 0.05, *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Co-Culture Assay, Gene Expression, Expressing, Cell Culture, Quantitative RT-PCR

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: Specificity of c10orf118 effect on HAS2 synthesis by NHDF. ( A ) Quantitative RT-PCR analysis of HAS2 expression by NHDF co-cultured with MCF-7 alone or in the presence of an anti-c10orf118 polyclonal antibody or an anti-α-actin antibody as negative control. Each bar represents mean ± S.D. of triplicates. * p < 0.05. ( B ) Quantitative RT-PCR analysis of HAS2 expression by NHDF in the presence of increasing concentrations of a recombinant c10orf118 peptide (Hr_Q71-211). Bars represent mean ± S.D. of three replicates. * p < 0.05.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Negative Control, Recombinant

Journal: Journal of Alzheimer's Disease

Article Title: Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

doi: 10.3233/jad-160804

Figure Lengend Snippet: Fig. 1. Immunohistochemical and immunofluorescence pictures demonstrate that HASs are widely expressed in the cortex and hippocampus of wild-type mice. A-C) In the hippocampus, Has1 not only localizes to the membrane of the cell body but also localizes to the membrane of the axon (Aa), Has2 is only present in the cytoplasm of the cells in the stratum pyramidale (Sp) and stratum oriens (So) of the hippocampus (Bb), and Has3 is only present in the cytoplasm of the cells in the stratum pyramidale (Sp) of the hippocampus (Cc). In the cortex, Has1 is also predominantly expressed in the cell membranes of the neurons (Aa’). Interestingly, however, we did not observe any localization of Has2 and Has3 in the axons in the cortex (Bb’ and Cc’). Has3 is the predominantly expressed HAS in blood vessels in the brain. Scale bar = 500 m. D) The distribution of HASs in the cortex were determined by immunofluorescence staining. NeuN is red. HASs are green. Scale bar = 10 m.

Article Snippet: Frozen sections were then166 treated with 5% goat serum/PBS for 30 min and167 subsequently incubated with Has1 (1:100, Sigma,168 SAB1302705), Has2 (1:100, Boster Biotechnology,169 BA3397-1), and Has3 (1:100; Santa Cruz, sc-66917)170 antibodies overnight at 4◦C.

Techniques: Immunohistochemical staining, Membrane, Staining

Journal: Journal of Alzheimer's Disease

Article Title: Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

doi: 10.3233/jad-160804

Figure Lengend Snippet: Fig. 3. Tau pathology promotes the production of short chain HA (<200 kDa) through regulating of HASs expression in hippocampus. A) Quantitative real-time PCR data showing the mRNA expression levels of Has1, Has2, and Has3 in the hippocampus of the TauP301S Tg mice and control mice (10-month-old). B-D) TauP301S inhibits the expression of Has1 and promotes Has3 expression in the hippocampus of TauP301S Tg mice. B) Immunoblot images (upper panel) and quantifications (lower panel) demonstrating that Has1 is decreased in hippocampus of the TauP301S Tg mice. C) Immunoblot images (upper panel) and quantifications (lower panel) illustrating that Has2 is not altered in the hippocampus of the TauP301S Tg mice. In contrast, the protein level of Has3 (D, left and right panels) is increased in the hippocampus of the TauP301S Tg mice. E) Immunoblot images (left panel) and quantifications (right panel) demonstrating that short chain HA (< 200 kDa) is increased in the hippocampus of TauP301S Tg mice (Tau P301S). The data are presented as relative fold changes from the normal controls (WT). Data are represented as means ± se.; n = 7 mice per group. *p < 0.05; ***p < 0.001. The p values were calculated using unpaired t-tests (A, middle and left panel; B, lower panel; C, lower panel; E, right panel) or unpaired t-tests with Welch’s corrections (A, left panel; D, right panel).

Article Snippet: Frozen sections were then166 treated with 5% goat serum/PBS for 30 min and167 subsequently incubated with Has1 (1:100, Sigma,168 SAB1302705), Has2 (1:100, Boster Biotechnology,169 BA3397-1), and Has3 (1:100; Santa Cruz, sc-66917)170 antibodies overnight at 4◦C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of Alzheimer's Disease

Article Title: Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

doi: 10.3233/jad-160804

Figure Lengend Snippet: Fig. 4. TauP301S expression inhibits the axonal-localization of Has1 in hippocampus. A, B) Immunohistochemical analyses (A) and immunofluorescence images (B) revealing the expressions and the distribution of Has1 in CA1 of the wild-type (WT) and TauP301S Tg (Tau P301S) mice (10-month-old). The enlarged regions of A showing the axonal-localization of Has1 in WT and TauP301S Tg mice. The lower panels of B are the enlarge Z section images of box 1 and 2 in the upper panel, showing the relative localization of Has1 (green) and NEFH (red) in the stratum radiatum of hippocampus. Scale bar = 50 m. C) Immunohistochemical analyses revealing the expressions of Has2 and Has3 in CA1 of WT and TauP301S Tg mice. Scale bar = 50 m.

Article Snippet: Frozen sections were then166 treated with 5% goat serum/PBS for 30 min and167 subsequently incubated with Has1 (1:100, Sigma,168 SAB1302705), Has2 (1:100, Boster Biotechnology,169 BA3397-1), and Has3 (1:100; Santa Cruz, sc-66917)170 antibodies overnight at 4◦C.

Techniques: Expressing, Immunohistochemical staining

Journal: Journal of Alzheimer's Disease

Article Title: Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

doi: 10.3233/jad-160804

Figure Lengend Snippet: Fig. 6. The expression of TauP301S did not affect the expressions of the HAS mRNAs in the cortex (10-month-old). Quantitative real-time PCR data showing the mRNA expression levels of Has1 (A), Has2 (B), and Has3 (C) in the cortex of the TauP301S Tg mice and control mice. The data are provided as the relative fold changes from the normal control. Data are represented as means ± se.; n = 7 mice per group. The p values were calculated using unpaired t-tests (A-C).

Article Snippet: Frozen sections were then166 treated with 5% goat serum/PBS for 30 min and167 subsequently incubated with Has1 (1:100, Sigma,168 SAB1302705), Has2 (1:100, Boster Biotechnology,169 BA3397-1), and Has3 (1:100; Santa Cruz, sc-66917)170 antibodies overnight at 4◦C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Alzheimer's Disease

Article Title: Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

doi: 10.3233/jad-160804

Figure Lengend Snippet: Fig. 7. The axonal-localization of Has1 in the cerebral cortex is affected by TauP301S in the TauP301S Tg mice. Immunoblot images (A-C, upper panels) and quantifications (A-C, lower panels) showing the expressions of Has1, Has2, and Has3 in the cerebral cortex of 10-month- old TauP301S Tg mice (Tau P301S) and control wild-type mice (WT). Data are represented as means ± se.; n = 7 mice per group. The p values were calculated using unpaired t-tests (A, B) and unpaired t-tests with Welch’s correction (C). D) Immunohistochemical images showing the distributions of Has 1 in 10-month-old WT and TauP301S Tg mice. The enlarged images (D, 1 and 2) show the axonal-localization of Has1 is inhibited by tau pathology. E-H) Immunofluorescence images (E, G) and quantifications (F, H) showing the Has1 is enriched in the cell body of neuronal cells in 10-month-old (E, F) and 3-week-old (G, H) TauP301S Tg mice. Data are represented as means ± se.; n = 20 cell bodies per group; the immunofluorescence intensity of Has1 in the cell body was normalized with the intensity of the surrounding area. ***p < 0.001. The p values were calculated using unpaired t-tests. Scale bar = 50 m.

Article Snippet: Frozen sections were then166 treated with 5% goat serum/PBS for 30 min and167 subsequently incubated with Has1 (1:100, Sigma,168 SAB1302705), Has2 (1:100, Boster Biotechnology,169 BA3397-1), and Has3 (1:100; Santa Cruz, sc-66917)170 antibodies overnight at 4◦C.

Techniques: Western Blot, Control, Immunohistochemical staining

Journal: Cartilage

Article Title: Elevated Glucose Levels Preserve Glucose Uptake, Hyaluronan Production, and Low Glutamate Release Following Interleukin-1β Stimulation of Differentiated Chondrocytes

doi: 10.1177/1947603518770256

Figure Lengend Snippet: (A, B, and D-I) Equine chondrocytes were differentiated into cartilage pellets in vitro and incubated with or without (basal) 5 ng/mL IL-1β for 72 hours in the presence of 5 mM or 25 mM glucose. (A) Cartilage pellets were sectioned and incubated with safranin O to analyze matrix content, followed by (B) scoring of safranin O color intensity, pellet size and morphology. The score was based on a scale of 1 to 5 for evaluating intensity of safranin O coloring, pellet size, and outer cell layer integrity of the pellet. (C) Equine cartilage explants were incubated in vitro with or without (basal) 10 ng/ml IL-1β for 24 days in the presence of 5 mM or 25 mM glucose and analyzed by hematoxylin and eosin staining. Modest fibrillation of the superficial layer of the articular cartilage in IL-1β-stimulated explants at 5 mM glucose is indicated by arrows. Scale bar: 100 μm. (D-I) mRNA expression of COL1A1, COL2A1, ACAN, COMP, MMP13, and HAS2, normalized to HPRT1 mRNA. Error bars indicate standard error of the mean (SEM), n = 4 (chondrocytes from 4 different horses). *P < 0.05.

Article Snippet: Gene Name Catalogue No. of Primer/Probe (Applied Biosystems) HPRT1 (Reference gene) Ec03470217_m1 SLC2A1/GLUT 1 EC03470363_m1 SGLT1 /SLC5A1 Ec03468144_m1 SLC2A4/GLUT 4 Ec03468109-m1 SLCA2 / GLUT 2 Ec03468195_m1 COL1A1 Ec03469676_m1 COL2A1 Ec03467378_m1 ACAN Ec03469667_m1 COMP Ec03468074_m1 MMP13 Ec03467797_m1 HAS2 Ec03467770_m1 TLR4 Ec03468994_m1 Open in a separate window Quantitative Real-Time Polymerase Chain Reaction Analysis

Techniques: In Vitro, Incubation, Staining, Expressing

Journal: Cartilage

Article Title: Elevated Glucose Levels Preserve Glucose Uptake, Hyaluronan Production, and Low Glutamate Release Following Interleukin-1β Stimulation of Differentiated Chondrocytes

doi: 10.1177/1947603518770256

Figure Lengend Snippet: Lactate secretion and relation of ENO1 with HAS2. Equine chondrocytes were differentiated into cartilage pellets in vitro and incubated with or without (basal) 5 ng/mL interleukin-1β (IL-1β) for 72 hours in the presence of 5 mM or 25 mM glucose. (A) Lactate secretion relative to 5 mM glucose in the basal state. Error bars indicate standard error of the mean (SEM), n = 4 (chondrocytes from 4 different horses). *P < 0.05. (B) STRING protein-protein interaction network analysis. Analysis settings were set for evidence-based interactions (indicated by lines between nodes) from textmining (yellow), gene neighborhood (green), experimentally determined (pink), gene fusions (red), curated databases (light blue), co-occurrence (dark blue) and co-expression (black). Colored nodes = query proteins and first shell interactions. Small nodes = proteins of unknown 3-dimensional (3D) structure. Large nodes = some 3D structure is known or predicted.

Article Snippet: Gene Name Catalogue No. of Primer/Probe (Applied Biosystems) HPRT1 (Reference gene) Ec03470217_m1 SLC2A1/GLUT 1 EC03470363_m1 SGLT1 /SLC5A1 Ec03468144_m1 SLC2A4/GLUT 4 Ec03468109-m1 SLCA2 / GLUT 2 Ec03468195_m1 COL1A1 Ec03469676_m1 COL2A1 Ec03467378_m1 ACAN Ec03469667_m1 COMP Ec03468074_m1 MMP13 Ec03467797_m1 HAS2 Ec03467770_m1 TLR4 Ec03468994_m1 Open in a separate window Quantitative Real-Time Polymerase Chain Reaction Analysis

Techniques: In Vitro, Incubation, Expressing

Journal: Cartilage

Article Title: Elevated Glucose Levels Preserve Glucose Uptake, Hyaluronan Production, and Low Glutamate Release Following Interleukin-1β Stimulation of Differentiated Chondrocytes

doi: 10.1177/1947603518770256

Figure Lengend Snippet: Hypothesis of the underlying mechanism for the effect of glucose during osteoarthritis. In conditions with osteoarthritis, inflammation causes degradation of cartilage components, including hyaluronan (HA), due to an insufficient supply of glucose and decreased glucose uptake in the chondrocyte. The decreased glucose uptake results in decreased expression of HAS2 and a lowering of the high molecular weight to low molecular weight (HMW:LMW) HA ratio. Reduced synthesis of HMW HA leads to a breakdown of the HA protective layer, exposing TLR-4 and enables interaction of TLR-4 with proinflammatory cytokines, thereby promoting inflammation. Furthermore, decreased HMW HA results in increased glutamate release, which in turn enhances inflammation. In addition, inflammatory cytokines, such as IL-1β, promote TLR-4 expression. The result is a self-sustained inflammatory cycle. By addition of glucose, the glucose uptake is sustained, resulting in maintained production of HMW HA by sustained HAS2 expression. Maintained HMW HA synthesis further leads to an intact protective HA layer as well as inhibition of glutamate release and maintained low expression of TLR-4. Maintained glucose uptake further enables a high glycolytic rate with increased lactate production. These anti-inflammatory actions interrupt the proinflammatory cycle and induces matrix repair.

Article Snippet: Gene Name Catalogue No. of Primer/Probe (Applied Biosystems) HPRT1 (Reference gene) Ec03470217_m1 SLC2A1/GLUT 1 EC03470363_m1 SGLT1 /SLC5A1 Ec03468144_m1 SLC2A4/GLUT 4 Ec03468109-m1 SLCA2 / GLUT 2 Ec03468195_m1 COL1A1 Ec03469676_m1 COL2A1 Ec03467378_m1 ACAN Ec03469667_m1 COMP Ec03468074_m1 MMP13 Ec03467797_m1 HAS2 Ec03467770_m1 TLR4 Ec03468994_m1 Open in a separate window Quantitative Real-Time Polymerase Chain Reaction Analysis

Techniques: Expressing, High Molecular Weight, Molecular Weight, Inhibition

Journal: Cartilage

Article Title: Elevated Glucose Levels Preserve Glucose Uptake, Hyaluronan Production, and Low Glutamate Release Following Interleukin-1β Stimulation of Differentiated Chondrocytes

doi: 10.1177/1947603518770256

Figure Lengend Snippet: Quantitative Real-Time Polymerase Chain Reaction Analysis

Article Snippet: Gene Name Catalogue No. of Primer/Probe (Applied Biosystems) HPRT1 (Reference gene) Ec03470217_m1 SLC2A1/GLUT 1 EC03470363_m1 SGLT1 /SLC5A1 Ec03468144_m1 SLC2A4/GLUT 4 Ec03468109-m1 SLCA2 / GLUT 2 Ec03468195_m1 COL1A1 Ec03469676_m1 COL2A1 Ec03467378_m1 ACAN Ec03469667_m1 COMP Ec03468074_m1 MMP13 Ec03467797_m1 HAS2 Ec03467770_m1 TLR4 Ec03468994_m1 Open in a separate window Quantitative Real-Time Polymerase Chain Reaction Analysis

Techniques: Real-time Polymerase Chain Reaction