Journal: ESC Heart Failure

Article Title: CT‐IGFBP‐4 as a novel prognostic biomarker in acute heart failure

doi: 10.1002/ehf2.12590

Figure Lengend Snippet: Correlation of N‐terminal pro brain natriuretic peptide (NT‐proBNP), CT‐IGFBP‐4, and C‐reactive protein (CRP) in a study cohort of patients with acute heart failure.

Article Snippet: The monoclonal antibodies IBP163, IBP182 conjugated with horseradish peroxidase (IBP182 HRP ), recombinant human CT‐IGFBP‐4, amino‐terminal fragment of IGFBP‐4 (NT‐IGFBP‐4), and IGFBP‐4 were obtained from HyTest Ltd, Turku, Finland.

Techniques:

Journal: ESC Heart Failure

Article Title: CT‐IGFBP‐4 as a novel prognostic biomarker in acute heart failure

doi: 10.1002/ehf2.12590

Figure Lengend Snippet: N‐terminal pro brain natriuretic peptide (NT‐proBNP) (A), CT‐IGFBP‐4 (B), and C‐reactive protein (CRP) (C) concentrations at admission in 1 year survivors and non‐survivors with acute heart failure. The central line represents median, box represents interquartile range, and whiskers represent 5 th and 95 th percentiles.

Article Snippet: The monoclonal antibodies IBP163, IBP182 conjugated with horseradish peroxidase (IBP182 HRP ), recombinant human CT‐IGFBP‐4, amino‐terminal fragment of IGFBP‐4 (NT‐IGFBP‐4), and IGFBP‐4 were obtained from HyTest Ltd, Turku, Finland.

Techniques:

Journal: ESC Heart Failure

Article Title: CT‐IGFBP‐4 as a novel prognostic biomarker in acute heart failure

doi: 10.1002/ehf2.12590

Figure Lengend Snippet: Receiver operator characteristic analysis of the clinical prediction model, N‐terminal pro brain natriuretic peptide (NT‐proBNP), CT‐IGFBP‐4, C‐reactive protein (CRP), and their combinations. Prediction of all‐cause mortality at 1 month (A) and 1 year (B) by NT‐proBNP, CT‐IGFBP‐4, CRP, and their combinations. P < 0.001 for all ROC curves compared with 0.5 curves.

Article Snippet: The monoclonal antibodies IBP163, IBP182 conjugated with horseradish peroxidase (IBP182 HRP ), recombinant human CT‐IGFBP‐4, amino‐terminal fragment of IGFBP‐4 (NT‐IGFBP‐4), and IGFBP‐4 were obtained from HyTest Ltd, Turku, Finland.

Techniques:

Journal: ESC Heart Failure

Article Title: CT‐IGFBP‐4 as a novel prognostic biomarker in acute heart failure

doi: 10.1002/ehf2.12590

Figure Lengend Snippet: Kaplan–Meier survival curve for patients according to N‐terminal pro brain natriuretic peptide (NT‐proBNP), CT‐IGFBP‐4, and C‐reactive protein levels. The patients are divided into two groups (A) or three groups (B) according to the NT‐proBNP (‘Increased’: ≥3078 pg/mL) and CT‐IGFBP‐4 (‘Increased’: ≥92.5 ng/mL) levels as indicated in the legends. Log‐rank P ‐values were <0.001 for all figures.

Article Snippet: The monoclonal antibodies IBP163, IBP182 conjugated with horseradish peroxidase (IBP182 HRP ), recombinant human CT‐IGFBP‐4, amino‐terminal fragment of IGFBP‐4 (NT‐IGFBP‐4), and IGFBP‐4 were obtained from HyTest Ltd, Turku, Finland.

Techniques:

Journal: Atherosclerosis Plus

Article Title: The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques

doi: 10.1016/j.athplu.2024.09.001

Figure Lengend Snippet: Expression pattern of components of the STC2 - PAPP-A - IGFBP4 - IGF1 axis. a. Carotid plaques stained by masson trichrome and immunohistochemistry for STC2, PAPP-A, IGFBP4, and IGF1R. Arrowheads point to examples of stained cells. b. Single-cell RNA sequencing data from Wirka et al., 2019, displayed as UMAPs showing annotated cell populations, and expression pattern of PAPP-A , STC2 , IGFBP4 , IGF1R , and IGF1 . SMC = smooth muscle cell; mSMC = modulated SMC; EC = endothelial cell.

Article Snippet: Recombinant human IGFBP4 from RnD Systems (Cat# 804-GB-025) and IGF1 from Austral Biologicals (Cat# GF-050-8) were pre-incubated in media/PBS? and added to all conditioned media from different plaque?-incubations (200 μL) in a final concentration of 1200 ng/mL (of both IGF1 and IGFBP4).

Techniques: Expressing, Staining, Immunohistochemistry, RNA Sequencing Assay

Journal: Atherosclerosis Plus

Article Title: The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques

doi: 10.1016/j.athplu.2024.09.001

Figure Lengend Snippet: Proteolytically active PAPP-A is present in atherosclerotic plaques. a. Study design: Plaque samples from carotid and femoral arteries were incubated in culture media for 24 h, and conditioned media was harvested for analyses. b-c. PAPP-A (b) and STC2 (c) concentration in conditioned media after 24 h of incubation quantified by ELISA. d. Principle of PAPP-A activity assay: Recombinant IGFBP4:IGF1 complex (32 kDa) is incubated with sample containing active PAPP-A resulting in proteolytic cleavage to 14 kDa IGFBP4 fragments. e. Conditioned media from each of the 20 plaque samples were incubated with recombinant IGFBP4:IGF1 for 0 or 24 h and analysed for IGFBP4 cleavage by Western blotting. In all samples, no IGFBP4 fragments are detected at the 0-h time point, but IGFBP4 fragments emerges after 24 h. No PAPP-A activity was detected in the media without tissue incubation (media 24 h). f. Quantitation of PAPP-A activity based on Western blotting. g. Correlation between PAPP-A concentration and -activity. h. Correlation between PAPP-A:STC2 molar ratio and PAPP-A activity. i. PAPP-A:STC2 molar ratio in conditioned media.

Article Snippet: Recombinant human IGFBP4 from RnD Systems (Cat# 804-GB-025) and IGF1 from Austral Biologicals (Cat# GF-050-8) were pre-incubated in media/PBS? and added to all conditioned media from different plaque?-incubations (200 μL) in a final concentration of 1200 ng/mL (of both IGF1 and IGFBP4).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Recombinant, Western Blot, Quantitation Assay

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: IGFBP-3 decreased lipogenesis and increased lipolysis. (A) 3T3-L1 adipocytes were treated with different doses of synthetic IGFBP-3 as indicated for 12 h. Lipolysis rate was determined using the Free Glycerol Reagent (Sigma). (B) 3T3-L1 adipocytes were treated with 14 C-labeled glucose together with different doses of synthetic IGFBP-3 for 12 h as indicated. Lipogenesis rate was measured by liquid scintillation counting. (C, D) 3T3-L1 adipocytes were treated with synthetic IGFBP-3 for 12 h, and TAG levels were determined by biochemical assay. Representative photos indicate the neutral lipids stained by BODIPY. * P <0.05 by two-tailed unpaired Student’s t test. BODIPY: Dipyrromethene Boron Difluoride; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; TAG: Triacylglycerol.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Labeling, Staining, Two Tailed Test, Binding Assay

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: IGFBP-3 decreased IGF/insulin signaling and dysregulated the expression of lipid metabolic genes. (A) 3T3-L1 adipocytes were treated with different doses of synthetic IGFBP-3 as indicated for 12 h. The expression of phosphorylation of Akt at Ser473 and Akt protein as well as phosphorylation of PKA substrate was determined using Western blot analysis. (B, C) RT-qPCR was performed to examine the mRNA level of lipogenic genes ( Acc1 , Acly , and Fasn ) and lipolytic genes ( Atgl and Hsl ). * P <0.05 vs . control. (D) 3T3-L1 adipocytes were treated with different doses of IGF-1 (0 ng/mL, 2 ng/mL, and 20 ng/mL) plus 1 µg/mL IGFBP-3 for 1 h. Western blot analysis was performed to determine the expression of p-Akt (Ser473) and Akt. (E) RT-qPCR analysis of the expression of Atgl and Fasn in 3T3-L1 adipocytes treated with IGF-1, IGFBP-3, or both for 12 h. Lipolysis was determined using the Free Glycerol Reagent (Sigma). BSA: Bovine serum albumin; Con: Control; FBS: Fetal bovine serum; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; mRNA: Messenger ribonucleic acid; N.S.: No significance; pAkt: Phosphorylated Akt; PKA: Protein kinase A; RT-qPCR: Real-time quantitative polymerase chain reaction.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: Cachectic tumor cells produced IGFBP-3 to suppress IGF signaling in adipocytes. (A, B) The expression of IGFBP-3 mRNA was analyzed in two independent published datasets (GSE16515 and GSE15471). (C) Western blot analysis on the expression of IGFBP-3 was performed in 3T3-L1 adipocytes as well as C26 and Capan-1 cells. (D, E) 3T3-L1 adipocytes were treated with conditioned medium (CM) from Capan1 or C26 cells with or without IGFBP-3 neutralizing antibody for 1 h. Western blot analysis of the expression of p-Akt (Ser473) and Akt was shown. (F, G) RT-qPCR was performed to determine the mRNA level of Atgl and Fasn in 3T3-L1 adipocytes treated as indicated for 12 h. * P <0.05 vs . control. Ab: Antibody; Ab-IGFBP-3: Antibody of IGFBP-3; Con: Control; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; mRNA: Messenger ribonucleic acid; N.S.: No significance; pAkt: Phosphorylated Akt; RT-qPCR: Real-time quantitative polymerase chain reaction.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Produced, Expressing, Western Blot, Quantitative RT-PCR, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: Cachectic tumor cells secreted IGFBP-3 to cause lipid loss in adipocytes. 3T3-L1 adipocytes were treated with CM from Capan1 or C26 cells without or with different doses of IGFBP-3 neutralizing antibody for 12 h. Lipolysis rate (A, E), lipogenesis rate (B, F), and TAG level (C, G) were examined. (D original magnification × 200, H original magnification ×200) The neutral lipids were stained with BODIPY and representative photos were indicated. Ab: Antibody; CM: Conditioned medium; Con: Control; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; TAG: Triacylglycerol.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Staining, Binding Assay

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: IGFBP-3 failed to affect the growth of cachectic cancer cells. (A, D) C26 cells were treated with different doses of IGFBP-3 or IGFBP-3 neutralizing antibody as indicated for 12 h. The expression of pAkt (Ser473) and Akt was shown using Western blot analysis. (B, E) Cell proliferation after IGFBP-3 treatment for three days was examined by MTT assay. (C, F) Cell morphology was observed by optical microscopy (C and F original magnification × 200). Ab: Antibody; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; MTT: Methyl thiazolyl tetrazolium assay; pAkt: Phosphorylated Akt.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Expressing, Western Blot, MTT Assay, Microscopy, Binding Assay, Methyl Thiazolyl Tetrazolium Assay

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: Gut tumors produced IGFBP-3 homolog to cause lipid loss in Drosophila . (A, B) Knockdown of ImpL2 or overexpression of ILP2 in yki -tumors at day 8 improved host abdominal fat in yki -gut Drosophila , and statistical analysis of abdomen bloating was performed ( n = 3, 5 flies/replicate) (A, [original magnification × 400]). Fat-body-specific reporter indicates depletion of this tissue in the abdomen of hosts in the presence of the tumor. Green indicates lipid droplets, which are the storage vesicles of the adipose tissue. (B) RT-qPCR was performed to determine the mRNA level of Fas and Bmm in gut tumors of Drosophila . (C) The bloating rates of wild-type yki , knock-down ImpL2 in yki -tumors and overexpression of ILP2 in yki -tumors, respectively. (D, E) The levels of TAG and trehalose were determined ( n = 3, 30 flies/group). * P <0.05 by two-tailed unpaired Student's t -test. IGFBP-3: Insulin-like growth factor binding protein-3; ILP2: Insulin-like peptide 2; ImpL2 -i: ImpL2 -RNAi; mRNA: Messenger ribonucleic acid; RT-qPCR: Real-time quantitative polymerase chain reaction; TAG: Triacylglycerol; w: Wild type.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Produced, Over Expression, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: IGFBP-3 was highly expressed in cancer tissues and serum of cachectic cancer. (A) Gene Expression Profiling Interactive Analysis (GEPIA) indicated IGFBP-3 was highly expressed in pancreatic cancer tissues ( P <0.05) and colorectal cancer tissues ( P >0.05) compared with normal tissues. (B, C) The association of the expression of IGFBP-3 with overall survival was analyzed using Kaplan–Meier (KM) plotter ( https://kmplot.com/analysis ) in both PAAD and COAD. The patients were divided by high and low IGFBP-3 expression levels using the auto-select best cutoff, and log-rank P -value was shown. (D) The serum level of IGFBP-3 in cachectic and non-cachectic colorectal cancer patients was determined by ELISA assay. This graph was generated using Graphpad prism 9. * P <0.05. COAD: Colonic adenocarcinoma; ELISA: Enzyme linked immunosorbent assay; HR: Hazard ratio; IGFBP-3: Insulin-like growth factor (IGF) binding protein-3; PAAD: Pancreatic adenocarcinoma; READ: Rectal adenocarcinoma.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Generated, Binding Assay

Journal: Chinese Medical Journal

Article Title: IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling

doi: 10.1097/CM9.0000000000002628

Figure Lengend Snippet: Schematic depiction of cachectic tumor cells-derived IGFBP-3 stimulates lipid loss by antagonizing IGF/insulin signaling. IGFBP-3: Insulin-like growth factor (IGF) binding protein-3.

Article Snippet: Synthetic IGFBP-3 (R&D, Minnesota, USA, 675-B3-025) and IGF-1 (R&D, 291-G1-200), IGFBP3 antibody (Santa Cruz, Dallas, Texas, USA, sc-9028), phosphorylated Akt (pAkt)-S473 antibody (Cell Signaling, Danvers, Massachusetts, USA, 9271), pAkt-T308 antibody (Cell Signaling, 13038), Akt antibody (Cell Signaling, 9272), phospho-protein kinases A (pPKA)-substrate (RRXS*/T*; Cell Signaling, 9624), α-Tubulin (Sigma, Merck KGaA, Darmstadt, Germany, T9026), and β-actin (Cell Signaling, 4970) were used in this study.

Techniques: Derivative Assay, Binding Assay

Journal: Scientific Reports

Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

doi: 10.1038/s41598-018-22855-z

Figure Lengend Snippet: IGFBP7 is identified as a osteogenic inductor in normal hMSCs. ( A ) Fluorescence images of antibody arrays obtained via laser scanning, showing spot signal intensities for TGFβ target genes, IGFBP7, TGFBI, FN and PAI-1. Duplicated spots for each protein are shown in the image. ( B ) Cell viability assay performed by CCK-8 kit at day 6 of cell culture under basal medium (BM) or osteogenesis induction medium (OIM). Results are normalized to those obtained from hMSCs without rIGFBP7 treatment under both BM or OIM cell culture conditions (n = 4). (C , D) ALP activity analysis in normal hMSCs after incubation with rIGFBP7, rFN, rTGFBI or rPAI-1 in BM or OIM during 6 days. Results are normalized to those obtained from hMSCs without rIGFBP7 treatment under BM (n = 4) (*p < 0.05, n = 4).

Article Snippet: Recombinant human IGFBP7, SerpinE1, Fibronectin and TGFBI (R&D Systems, Cat N°: 1334-B7, 1786-PI, 4305-FN and 3409-GB respectively) were independently added to cell culture at the indicated final concentrations and incubated for 6 days.

Techniques: Fluorescence, Viability Assay, CCK-8 Assay, Cell Culture, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis

doi: 10.1038/s41598-018-22855-z

Figure Lengend Snippet: IGFBP7 expression is essential for hMSCs survival during early osteogenesis. ( A ) Scheme of the workflow carried out to study the effects of IGFBP7 silencing during hMSCs early osteogenesis. Two rounds of siRNA were performed to ensure the continuous silencing of IGFBP7 during the 6 days (d) of osteogenesis differentiation. The effects of IGFBP7 silencing were assessed after 2 and 6 days of osteogenesis of hMSCs. ( B ) IGFBP7 mRNA expression was studied by RT-PCR after 2 and 6 days of osteogenesis induction. IGFBP7 expression in siIGFBP7 hMSCs was normalized to Gapdh and fold induction was calculated in reference to hMSCs transfected with siNT (n = 5). In parallel, the expression of IGFBP7 was studied in non-transfected hMSCs. IGFBP7 fold induction was calculated in reference to hMSCs without differentiation induction at day 2 of cell culture (n = 5). ( C ) Cell viability after IGFBP7 silencing was assessed after 6 days of osteogenic differentiation. Transfected hMSCs viability was normalized to that observed in non-transfected hMSCs (n = 5). ( D ) Alkaline phosphatase activity was assessed at day 6 of osteogenesis after siRNA. Results are normalized to the alkaline phosphatase activity of hMSCs transfected with siNT (n = 5). Negative and positive controls of osteogenesis, without transfecting are shown. ( E ) Runx2 expression was assessed at day 2 and 6 of osteogenesis after siRNA and was normalized to Gapdh. Runx2 fold induction was calculated in reference to Runx2 expression of hMSCs transfected with siNT at day 2. As a control, the expression of Runx2 was also studied in non-transfected hMSCs at day 2 and 6 of cell culture. In this case, Runx2 fold induction is calculated in reference to Runx2 expression in hMSCs without differentiation at day 2 of cell culture (n = 5). Results are expressed as mean ± SD, *** indicates p < 0.001; ** indicates p < 0.01 and * indicates p < 0.05.

Article Snippet: Recombinant human IGFBP7, SerpinE1, Fibronectin and TGFBI (R&D Systems, Cat N°: 1334-B7, 1786-PI, 4305-FN and 3409-GB respectively) were independently added to cell culture at the indicated final concentrations and incubated for 6 days.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Activity Assay, Control