Journal: International journal of molecular sciences

Article Title: Influence of Anticoagulants on the Dissociation of Cardiac Troponin Complex in Blood Samples.

doi: 10.3390/ijms25168919

Figure Lengend Snippet: Figure 3. Detection of the ITC-complex dissociation products in the GF profiles before and after 3 h of incubation at 37 ◦C in different matrices. The ITC-complex spiked in citrate plasma (A,B), heparin plasma (C,D), EDTA plasma (E,F) and serum (G,H), and troponin immunoreactivity in the GF fractions was measured via FIA using the Tcom8-TnT7E7 (specific to the ITC-complex, black), TnI84-TnC7B9 (specific to the IC-complex, red), TnC12G3-TnC7B9 (specific to the IC-complex and free TnC, blue), and TnT155-TnT7E7 (specific to free cTnT, green) assays.

Article Snippet: Anti-TnI monoclonal antibodies (mAbs): TnI84 (specific to amino acid residues (aar) 117–126); anti-TnT mAbs: TnT329 (aar 119–138), TnT7E7 (aar 223–242), TnT155 (aar 263–281), anti-TnC mAbs TnC12G3, TnC7B9, and mAb Tcom8 specific to the cardiac ICand ITCcomplexes were obtainedfrom HyTest (Turku, Finland).

Techniques: Incubation, Clinical Proteomics

Journal: International journal of molecular sciences

Article Title: Influence of Anticoagulants on the Dissociation of Cardiac Troponin Complex in Blood Samples.

doi: 10.3390/ijms25168919

Figure Lengend Snippet: Figure 4. Detection of endogenous troponin complex dissociation products from GF. Citrate plasma sample (A,B) and heparin plasma (C,D) from a patient with AMI were incubated at 37 ◦C, and troponin immunoreactivity in the GF fractions was measured by FIA using the Tcom8-TnT7E7 (specific to the ITC-complex, black), TnI84-TnC7B9 (specific to the IC-complex, red), TnC12G3- TnC7B9 (specific to the IC-complex and free TnC, blue) and TnT155-TnT7E7 (specific to free cTnT, green) assays. Solid grey line–protein profile (mAU280).

Article Snippet: Anti-TnI monoclonal antibodies (mAbs): TnI84 (specific to amino acid residues (aar) 117–126); anti-TnT mAbs: TnT329 (aar 119–138), TnT7E7 (aar 223–242), TnT155 (aar 263–281), anti-TnC mAbs TnC12G3, TnC7B9, and mAb Tcom8 specific to the cardiac ICand ITCcomplexes were obtainedfrom HyTest (Turku, Finland).

Techniques: Clinical Proteomics, Incubation

Journal: American Journal of Cancer Research

Article Title: Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori -activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

doi:

Figure Lengend Snippet: Pro-migratory and pro-metastatic potential of l.t.EMT+RGM1 cells is associated with the activation of HGF/TNC/Integrin-β1 pathway. RT-PCR analysis of the adhesion and motility related mRNA expression (18S, cMet, HGF, EGFR and TNC) in l.t.EMT-RGM1, l.t.EMT+RGM1 and original RGM1 cells and the ratio of selected genes over 18S mRNA (A). Elisa analysis of HGF content in the secretome. The cells were transferred to DMEM+10% FBS and allowed for 48 hrs secretion. The analysis showed that only l.t.EMT+RGM1 cells were able to secrete HGF (B). Western Blot analysis of c-Met, p-EGFR, integrin-β1 and TNC protein expression in total cellular proteins isolated from l.t.EMT-RGM1, l.t.EMT+RGM1 and the original RGM1 cells and the semi-quantitative densitometry analysis of the ratio of selected proteins over GAPDH showing the upregulation of c-Met, p-EGFR, Integrin-β1 and TNC proteins in l.t.EMT+RGM1 cells. 10 µg and in the case of EGFR and TNC 25 µg of total cellular proteins were loaded per each lane (C). Results are mean ± SEM of four to six independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control RGM1 value. Hash (#) indicates a significant change (P<0.05) as compared to l.t.EMT-RGM1 value.

Article Snippet: The primary antibodies used: anti-GAPDH (D16H11 Cell Signaling), anti-Snail (ab180714, Abcam, Cambridge, UK), anti-Twist (ab50887 Abcam, Cambridge, UK), anti-Cytokeratine19 (GTX112666 Genetex, Irvine, CA, USA), anti-TNC (GTX17605 Genetex, Irvine, CA, USA), anti-β1-integrin (GTX128839 Genetex, Irvine, CA, USA), anti-c-Met (25869-1-AP ProteinTech, Manchester, UK) and anti-p-EGFR (2231S, Cell Signaling, Danvers, MA, USA) and anti-EGFR (ab52894, Abcam, Cambridge, UK).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation

Journal: American Journal of Cancer Research

Article Title: Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori -activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

doi:

Figure Lengend Snippet: Hp-hAGF secretome induces increased invasiveness of cancer cells. This phenomenon in adenocarcinoma cells is related to cMet/HGF/Integrin-β1/TNC and EGFR signaling. Geltrex invasion assay of HT29 and AGS cells towards culture medium, hGF and Hp-hAGF supernatants and in the case of AGS cells towards Hp-hAGF secretome with the addition of tyrphostin A46 (0.1 mM) showing enhanced invasiveness towards Hp-hAGF supernatant and the decrease after A46 (A). Fluorescent microscopy of transmigrated AGS cells attached to the bottom side of the geltrex/transwell membrane stained with bisbenzimide (Hoechst 33342) (B). The phase contrast microscopy showing HT29 and AGS cells that transmigrated through invasion chambers towards Hp-hAGF supernatant. Three independent experiments were performed for each condition (C). RT-PCR analysis of the expression of 18S, Fap, MMP 2, 3, 9 and mRNA in HT29 (D) and AGS (E) cells cultured in culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells in Hp-hAGF secretome with addition of tyrphostin A46 (0.1 mM) and the ratio of selected genes over 18S mRNA showing their strong upregulation in Hp-hAGF secretome. Phase contrast microscopy of Geltrex metalloproteinase activity assay (1:1) of HT29 (F) and AGS (G) flooded with culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells with Hp-hAGF secretome with the addition of A46 (0.1 mM), showing enhanced ability of cells flooded with Hp-hAGF supernatant to cross basement membrane components already after 24 hrs. RT-PCR analysis of the mRNA expression for 18S, cMet, HGF, EGFR and Integrin-β1 in AGS cells cultured in control medium, hGF, Hp-hAGF and in Hp-hAGF secretome with the addition of A46 (0.1 mM) and the ratio of selected genes over 18S mRNA (H). Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control cell value. Hash (#) indicates a significant change (P<0.05) as compared to the value of cells grown in GF supernatant.

Article Snippet: The primary antibodies used: anti-GAPDH (D16H11 Cell Signaling), anti-Snail (ab180714, Abcam, Cambridge, UK), anti-Twist (ab50887 Abcam, Cambridge, UK), anti-Cytokeratine19 (GTX112666 Genetex, Irvine, CA, USA), anti-TNC (GTX17605 Genetex, Irvine, CA, USA), anti-β1-integrin (GTX128839 Genetex, Irvine, CA, USA), anti-c-Met (25869-1-AP ProteinTech, Manchester, UK) and anti-p-EGFR (2231S, Cell Signaling, Danvers, MA, USA) and anti-EGFR (ab52894, Abcam, Cambridge, UK).

Techniques: Invasion Assay, Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Activity Assay