Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: CRP promotes osteo-/chondrogenic transdifferentiation of HAoSMCs. ( A ) Scatter dot plots and arithmetic means ± SEM (n=8; arbitrary units, a.u.) of CBFA1 relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( B ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM (n=10; a.u.) of normalized CBFA1/GAPDH protein ratio in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( C ) Scatter dot plots and arithmetic means ± SEM (n=8; a.u.) of ALPL relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( D ) Scatter dot plots and arithmetic means ± SEM (n=6, U/mg protein) of ALPL activity in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( E ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM (n=10; a.u.) of normalized αSMA/GAPDH protein ratio in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. *(p<0.05), **(p<0.01) significant vs. control HAoSMCs.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Expressing, Control, Recombinant, Western Blot, Activity Assay

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: CRP augments phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. ( A, B ) Scatter dot plots and arithmetic means ± SEM (n=6; arbitrary units, a.u.) of CBFA1 ( A ) and ALPL ( B ) relative mRNA expression in HAoSMCs treated with control (CTR) or β-glycerophosphate (Pi) without and with 10 µg/ml recombinant human CRP. ( C ) Representative original images (n=4) showing Alizarin red staining in HAoSMCs treated with control (CTR) or calcification medium (Calc.) without and with 10 µg/ml recombinant human CRP. The calcified areas are shown as red staining. ( D ) Scatter dot plots and arithmetic means ± SEM (n=6; µg/mg protein) of calcium content in HAoSMCs treated with control (CTR) or calcification medium (Calc.) without and with 10 µg/ml recombinant human CRP. **(p<0.01), ***(p<0.001) significant vs. control HAoSMCs; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. HAoSMCs treated with Pi/Calc. alone.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Expressing, Control, Recombinant, Staining

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: CRP increases cellular oxidative stress and oxidative stress-downstream signaling in HAoSMCs. ( A, B ) Scatter dot plots and arithmetic means ± SEM (n=8; arbitrary units, a.u.) of NOX4 ( A ) and CYBA ( B ) relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( C ) Scatter dot plots and arithmetic means ± SEM (n=6; a.u.) of normalized total antioxidant capacity of HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( D ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM (n=5; a.u.) of normalized phospho-p38/total p38/GAPDH protein ratio in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( E-G ) Scatter dot plots and arithmetic means ± SEM (n=8; a.u.) of MMP2 ( E ), MMP9 ( F ) and PAI1 ( G ) relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. ( H ) Representative original Western blots and scatter dot plots and arithmetic means ± SEM (n=7; a.u.) of normalized caspase 3/GAPDH and cleaved caspase 3/GAPDH protein ratio in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP. *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. control HAoSMCs.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Expressing, Control, Recombinant, Western Blot

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: Antioxidants suppress CRP-induced osteogenic signaling in HAoSMCs. ( A-E ) Scatter dot plots and arithmetic means ± SEM (n=6; arbitrary units, a.u.) of MMP2 ( A ), MMP9 ( B ), PAI1 ( C ), CBFA1 ( D ) and ALPL ( E ) relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP without and with 10 µM TEMPOL or 10 µM TIRON. *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. control HAoSMCs; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. HAoSMCs treated with CRP alone.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Expressing, Control, Recombinant

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: Inhibition of p38 MAPK blunts CRP-induced osteogenic signaling in HAoSMCs. ( A-E ) Scatter dot plots and arithmetic means ± SEM (n=5; arbitrary units, a.u.) of MMP2 ( A ), MMP9 ( B ), PAI1 ( C ), CBFA1 ( D ) and ALPL ( E ) relative mRNA expression in HAoSMCs treated with control (CTR) or 10 µg/ml recombinant human CRP without and with 10 µM p38 MAPK inhibitor SB203580 (SB). **(p<0.01), ***(p<0.001) significant vs. control HAoSMCs; ††(p<0.01) significant vs. HAoSMCs treated with CRP alone.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Inhibition, Expressing, Control, Recombinant

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: Antioxidants or p38 MAPK inhibition reduce calcification of HAoSMCs promoted by CRP during pro-calcific conditions. Scatter dot plots and arithmetic means ± SEM (n=6; µg/mg protein) of calcium content in HAoSMCs treated with control (CTR) or calcification medium together with 10 µg/ml recombinant human CRP (Calc.+CRP) and without and with additional treatment with 10 µM TEMPOL, 10 µM TIRON or 10 µM p38 MAPK inhibitor SB203580 (SB). *(p<0.05), **(p<0.01) significant vs. control HAoSMCs; †(p<0.05) significant vs. HAoSMCs treated with Calc.+CRP alone.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Inhibition, Control, Recombinant

Journal: Aging (Albany NY)

Article Title: Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells

doi: 10.18632/aging.102130

Figure Lengend Snippet: Silencing of FCGR2A inhibits CRP-induced osteogenic signaling and calcification of HAoSMCs. ( A-E ) Scatter dot plots and arithmetic means ± SEM (n=6; arbitrary units, a.u.) of FCGR2A ( A ), NOX4 ( B ), CYBA ( C ), CBFA1 ( D ) and ALPL ( E ) relative mRNA expression in HAoSMCs silenced with negative control siRNA (Neg.si) or FCGR2A siRNA (FR2Asi) and treated with control or 10 µg/ml recombinant human CRP. ( F ) Scatter dot plots and arithmetic means ± SEM (n=6; µg/mg protein) of calcium content in HAoSMCs silenced with negative control siRNA (Neg.si) or FCGR2A siRNA (FR2Asi) and treated with control or calcification medium together with 10 µg/ml recombinant human CRP (Calc.+CRP). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. Neg.si silenced HAoSMCs; ††(p<0.01), †††(p<0.001) significant vs. Neg.si silenced and CRP/Calc.+CRP treated HAoSMCs.

Article Snippet: At confluence, HAoSMCs were split into 6-well plates (2x10 5 cells/well) and allowed to attach for 24 hours prior to treatment for the indicated times with 10 μg/ml recombinant human CRP protein (R&D Systems) [ , ], 10 μM TEMPOL (4-hydroxy-TEMPO, stock in DMSO; Sigma-Aldrich), 10 μM TIRON (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate; Sigma-Aldrich) [ , ], 10 μM p38 MAPK inhibitor SB203580 (stock in DMSO; Cayman Chemical) [ ] or 100 nM aldosterone (stock in DMSO; Sigma-Aldrich) [ , ].

Techniques: Expressing, Negative Control, Control, Recombinant

Journal: International journal of veterinary science and medicine

Article Title: Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP.

doi: 10.1080/23144599.2023.2247250

Figure Lengend Snippet: Figure 2. Schematic representation of FIA cCRP. FIA cCRP was designed as a medical device for in vitro diagnostic purposes that utilizes immunochromatography to quantitatively detect cCRP in dog serum or plasma, aiding in the diagnosis of inflammation. The device works by binding cCRP-specific antibodies to fluorescent particles such as europium in the fluorescent conjugate, as well as cCRP-specific antibodies bound to the nitrocellulose membrane test line. The fluorescent conjugate reacts primarily with cCRP in the sample to form an antigen-antibody complex, which moves along the membrane by capillary reaction. The complex then reacts with secondary cCRP antibodies to form an antigen-antibody complex that becomes immobilized on the line zone, providing a fluorescent signal strength that corresponds to the cCRP concentration present in the sample. The device is used with a dedicated analyser to measure cCRP levels. FIA, fluorescent immunoassay; cCRP, canine c-reactive protein.

Article Snippet: All assays used a recombinant cCRP (Cat. 8CC5, Hytest, Finland) as standard.

Techniques: In Vitro, Diagnostic Assay, Clinical Proteomics, Biomarker Discovery, Binding Assay, Membrane, Concentration Assay

Journal: International journal of veterinary science and medicine

Article Title: Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP.

doi: 10.1080/23144599.2023.2247250

Figure Lengend Snippet: Figure 3. Linearity curve of measured value for cCRP using FIA cCRP. R2 was 0.9977. cCRP, canine c-reactive protein; Conc., concentration; FIA, fluorescent immunoassay.

Article Snippet: All assays used a recombinant cCRP (Cat. 8CC5, Hytest, Finland) as standard.

Techniques: Concentration Assay

Journal: International journal of veterinary science and medicine

Article Title: Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP.

doi: 10.1080/23144599.2023.2247250

Figure Lengend Snippet: Figure 4. Correlation between the FIA cCRP and ELISA. Correlation coefficient (Pearson’s ρ) was 0.94 (p < 0.001; n = 21). FIA, fluorescent immunoassay; cCRP, canine c-reactive protein; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: All assays used a recombinant cCRP (Cat. 8CC5, Hytest, Finland) as standard.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International journal of veterinary science and medicine

Article Title: Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP.

doi: 10.1080/23144599.2023.2247250

Figure Lengend Snippet: Figure 5. Bland–Altman difference plot. Bias was 15.9 (SD ±27.4, 95% limits of agreement between − 37.77 and 69.58). SD, standard deviation; FIA, fluorescent immunoassay; cCRP, canine c-reactive protein; ELISA, enzyme-linked immu nosorbent assay.

Article Snippet: All assays used a recombinant cCRP (Cat. 8CC5, Hytest, Finland) as standard.

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Microbial Cell Factories

Article Title: Production of single chain Fab (scFab) fragments in Bacillus megaterium

doi: 10.1186/1475-2859-6-38

Figure Lengend Snippet: Purification and analysis of anti-CRP LA13-IIE3 scFvs. A Westernblot and immunostain of the combined supernatant of 3 independent productions, wash fractions and elution fractions of the IMAC purification. Samples were separated by reducing SDS-PAGE (12%) and detected as described in figure 3. B Antigen binding ELISA of purified anti-CRP scFv produced in B. megaterium or E. coli . Antigens: 100 ng/well CRP or 100 ng/well control protein BSA, detection as described in figure 2.

Article Snippet: Maxisorb MTPs (Nunc, Wiesbaden, Germany) were coated with either 1 μg hen egg white lysozyme, 100 ng CRP (BiosPacific, Emeryville, California) or BSA as control protein in 100 μL PBS per well overnight at 4°C.

Techniques: Purification, SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay, Produced, Control