Journal: Scientific Reports

Article Title: Adenosine deaminase modulates metabolic remodeling and orchestrates joint destruction in rheumatoid arthritis

doi: 10.1038/s41598-021-94607-5

Figure Lengend Snippet: Showing ADA Activity and Cytokine levels in Rheumatoid arthritis patients. ( a ) ADA activity in RA patients representing group with Low ADA and High ADA when compared to healthy controls. ( b ) ADA activity of two isoenzymes ADA 1 and ADA 2 (*representing a p value of ≤ 0.05) ( c ) Levels of Cytokines in the serum of RA patients. ( d ) One-way ANOVA analysis of Cytokines and ADA levels in healthy controls, Low ADA and High ADA RA patients. (p-value of ≤ 0.05 is considered significant). All the plots were made using Origin 2020.

Article Snippet: TNFα (Cat. No. CHC1753), TGFβ (Cat. No. CHC1683), IFNγ (Cat. No. CHC1233), and IL-10 (Cat. No. CHC1323) (Invitrogen Cytoset Antibody Pair Kit , California, USA) and IL-6 (Cat. No. 900 M16) and sRANKL (Cat. No. 900 M142) (Peprotech Mini ABTS ELISA Kit, New Jersey, USA) were estimated using manufacturers’ protocols and calculated by the 4-parameter curve-fit method.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Adenosine deaminase modulates metabolic remodeling and orchestrates joint destruction in rheumatoid arthritis

doi: 10.1038/s41598-021-94607-5

Figure Lengend Snippet: ADA is involved in joint remodeling in rheumatoid arthritis—in vitro studies to investigate the role of ADA in joint remodeling in the affected joint. ( a ) Increased proliferation of Synoviocytes, ( b ) Decreased cell viability in primary chondrocytes. ( c ) Increased Osteoclast differentiation of macrophages and ( d ) qPCR analysis showing the expression of osteoclast markers after 72 h exposure to ADA. ( e ) Increased cytokine levels in the spent medium after 72 exposure to ADA. ( f ) P2Y 2 , P2Y 4 , and P2Y 6 receptor expression in macrophages after 72 h of ADA exposure. (showing *representing a p value of ≤ 0.05, and **representing a p value of ≤ 0.005). ( g ) Increased ROS in macrophages after ADA exposure (p values of ADA to CTRL on all time points are ≤ 0.005). All the plots were made using Origin 2020.

Article Snippet: TNFα (Cat. No. CHC1753), TGFβ (Cat. No. CHC1683), IFNγ (Cat. No. CHC1233), and IL-10 (Cat. No. CHC1323) (Invitrogen Cytoset Antibody Pair Kit , California, USA) and IL-6 (Cat. No. 900 M16) and sRANKL (Cat. No. 900 M142) (Peprotech Mini ABTS ELISA Kit, New Jersey, USA) were estimated using manufacturers’ protocols and calculated by the 4-parameter curve-fit method.

Techniques: In Vitro, Expressing

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: The PMA dose response. Cultured human glial cells (T98G) were incubated for 12 hours with the indicated concentration of PMA and then secreted NGF titers were determined using an NGF-specific ELISA. All assays were performed in at least triplicate. Non-linear regression fitting indicates the half-maximal activity of PMA occurs at 6.6 nM (R 2 = 0.95). Notably, a suppression of NGF titer is observed at an elevated PMA concentration (10 μM), likely reflective of phorbol-ester cytotoxicity [ , ].

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Cell Culture, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: Effects of CREB Inhibitor 217505 on PMA induction of NGF. A) An alamar blue based colorimetric assay was used to determine the viability of cultured human glial cells exposed to vehicle (DMSO) or 10 nM PMA coupled with 1 μM of 217505, as indicated. Percent viability is relative to the culture exposed to vehicle alone. CREB inhibitor 217505 has no effect on glial cell viability, either alone (not shown) or in combination with 10 nM PMA. B) Conditioned media obtained from human glial cell cultures exposed to either vehicle (DMSO), 10 nM PMA, or 10 nM PMA+1 μM 217505 were assayed for NGF content using an NGF-specific ELISA. The glial cells were preincubated with 217505 for 20 mins, then PMA was added and NGF titers were determined after a 4 hr incubation. 217505 has no significant effect on PMA-induced glial cell NGF secretion within four hours. C) Quantitative PCR assessment of NGF transcript in human glial cell cultures treated with vehicle (DMSO), 10 nM PMA, or 10 nM PMA coupled with 1 μM of 217505, as indicated. The cells were treated as described in B). 217505 significantly reduces PMA-induced NGF gene expression (n = 6). As a side note, 217505 is insoluble in media at concentrations above 1 μM. All graphs are expressed as Mean + SD.

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Colorimetric Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: Effects of bryostatin-1 on NGF gene expression and secretion. A) T98G cells were treated with Bryostatin (10 nM), PMA (10 nM) or vehicle alone for four hours. After four hours, cell viability was measured by resazurin assay. Percent viability is relative to the culture exposed to vehicle alone. While PMA demonstrates some measurable toxicity, bryostatin-1 has no detrimental effect on cell viability (p > 0.05, n = 6). Both PMA and bryostatin-1 were assayed at 10 nM concentration. B) Conditioned media obtained from human glial cell cultures exposed to either vehicle (DMSO), bryostatin-1, or PMA were assayed for NGF content using the NGF-specific ELISA. The PKC agonist bryostatin-1 induces NGF secretion from the cultured glial cells. NGF titers were determined after 4 hr. incubation, with both PMA and bryostatin-1 at 10 nM concentration. Both agonists demonstrated statistically significant induction of NGF secretion (n = 6). C) Quantitative PCR assessment of NGF transcript in human glial cell cultures treated with vehicle (DMSO) and increasing concentrations of bryostatin-1. Non-linear regression fitting a dose-response curve indicates the half-maximal activity of bryostatin-1 gene induction occurs at 170 pm (R 2 = 0.91). All graphs are expressed as Mean + SD.

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Gene Expression, Resazurin Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: Effects of the MEK inhibitor UO126 on PMA induction of NGF gene expression and secretion. A) A colorimetric assay with alamar blue was used to determine the viability of cultured glial cells exposed to vehicle (DMSO) or 10 nM PMA in conjunction with various concentrations of UO126, as indicated. Percent viability is relative to the culture exposed to vehicle. UO126 alone has little effect on glial cell viability (n = 6). B) Conditioned media obtained from human glial cell cultures exposed to either vehicle (DMSO), 10 nM PMA, or 10 nM PMA+10 nM UO126 were assayed for NGF content using the NGF-specific ELISA. The glial cells were preincubated with UO126 for 20 mins, then PMA was added and NGF titers were determined after a 4 hr incubation. UO126 eliminates PMA-induced glial cell NGF secretion (n = 6). C) Quantitative PCR assessment of NGF transcript in human glial cell cultures treated with vehicle (DMSO), 10 nM PMA, or 10 nM PMA coupled with various concentrations of UO126, as indicated. UO126 reduces PMA-induced NGF gene expression in a dose-dependent manner. Statistical significance (t-test PMA versus vehicle, PMA versus PMA + Inhibitor) indicated when applicable (n = 6). Non-linear regression fitting a dose-response curve indicates the half-maximal inhibitory activity of UO126 occurs at 401 nM (R 2 = .803). All graphs are expressed as Mean + SD.

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Gene Expression, Colorimetric Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: Effects of the ERK inhibitor FR180204 on PMA induction of NGF gene expression and secretion. A) An alamar blue assay was used to determine the viability of cultured human glial cells exposed to vehicle (DMSO) or 10 nM PMA in conjunction with various concentrations of FR180204, as indicated. Percent viability is relative to the culture exposed to vehicle. FR180204 alone has no substantial effect on glial cell viability (n = 6). B) Conditioned media obtained from human glial cell cultures exposed to either vehicle (DMSO), 10 nM PMA, or 10 nM PMA+10 nM FR180204 were assayed for NGF content using the NGF-specific ELISA. The glial cells were preincubated with FR180204 for 20 mins, then PMA was added and NGF titers were determined after a 4 hr incubation. FR180204 reduces PMA-induced glial cell NGF secretion (n = 6). C) Quantitative PCR assessment of NGF transcript in human glial cell cultures treated with vehicle (DMSO), 10 nM PMA, or 10 nM PMA coupled with various concentrations of FR180204, as indicated. FR180204 reduces PMA-induced NGF gene expression in a dose-dependent manner. Statistical significance (t-test PMA versus vehicle, PMA versus PMA + Inhibitor) indicated when applicable (n = 6). Non-linear regression fitting a dose-response curve indicates the half-maximal inhibitory activity of FR180204 occurs at 1.90 uM (R 2 = 0.828). All graphs are expressed as Mean + SD.

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Gene Expression, Alamar Blue Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Heliyon

Article Title: Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

doi: 10.1016/j.heliyon.2018.e00617

Figure Lengend Snippet: Effects of the p90RSK Inhibitor BI-D1870 on PMA induction of NGF gene expression and secretion. A) An alamar blue colorimetric assay was used to determine the viability of cultured human glial cells exposed to vehicle (DMSO) or 10 nM PMA in conjunction with various concentrations of BI-D1870, as indicated. Percent viability is relative to the culture exposed to vehicle. BI-D1870 alone has no significant effect on glial cell viability (n = 6). B) Conditioned media obtained from human glial cell cultures exposed to either vehicle (DMSO), 10 nM PMA, or 10 nM PMA+10 nM BI-D1870 were assayed for NGF content using the NGF-specific ELISA. The glial cells were preincubated with BI-D1870 for 20 mins, then PMA was added and NGF titers were determined after a 4 hr incubation. BI-D1870 reduces PMA-induced glial cell NGF secretion (n = 6). C) Quantitative PCR assessment of NGF transcript in human glial cell cultures treated with vehicle (DMSO), 10 nM PMA, or 10 nM PMA coupled with various concentrations of BI-D1870, as indicated. Statistical significance (t-test PMA versus vehicle, PMA versus PMA + Inhibitor) indicated when applicable (n = 6). BI-D1870 has no significant effect on PMA induced NGF gene expression. All graphs are expressed as Mean + SD.

Article Snippet: After assay completion, the conditioned media was harvested and NGF titers were determined using a Human b-NGF ELISA Development Kit (Peprotech, Rocky Hill, NJ USA) per the manufacturer's instructions.

Techniques: Gene Expression, Colorimetric Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction