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Abcam igf 1 receptor blocking antibody

A . VEGF concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, Fibro+Myo, cCSC4A+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . TGF-β1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. all. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. C . <t>IGF-1</t> concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, †P<0.05 vs. Fibro+Myo, cCSC1A+Myo, cCSC3C+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. D. IGF-1 concentration correlated with cardiomyocyte contractility. Correlation was determined using Pearson Product Moment Correlation coefficient.

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1) Product Images from "c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling"

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014297

Figure Legend Snippet: A . VEGF concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, Fibro+Myo, cCSC4A+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . TGF-β1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. all. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. C . IGF-1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, †P<0.05 vs. Fibro+Myo, cCSC1A+Myo, cCSC3C+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. D. IGF-1 concentration correlated with cardiomyocyte contractility. Correlation was determined using Pearson Product Moment Correlation coefficient.

Techniques Used: Concentration Assay, Cell Culture, Expressing

A–C . Representative confocal microscopy images showing GFP pos (green) GATA-4 high c-kit pos cCSCs (cCSC4A+Myo) differentiated into the cardiomyocyte lineage (α-sarcomeric actin pos ; red) when co-cultured with adult rat cardiomyocytes (α-sarcomeric actin pos ; red) over 14 days (B). Note the lack of cardiomyogenic differentiation of GFP pos (green) GATA-4 high c-kit pos cCSCs at 3 days (cCSC4A+Myo; A) and GFP pos (green) GATA-4 low c-kit pos cCSCs at 14 days (cCSC3C+Myo; C). Bar = 20 µm. D . The percent number of GFP pos α-sarcomeric actin pos cells at 3, 7 and 14 days in vitro (DIV) following co-culture of GFP pos GATA-4 low expressing c-kit pos cCSCs with cardiomyocytes (cCSC3C+Myo), GFP pos GATA-4 high c-kit pos cCSCs with cardiomyocytes (cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. cCSC3C+Myo. ** vs. cCSC3C alone. † vs. cCSC4A alone. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. E . The percent number of GFP pos α-sarcomeric actin pos cCSC-derived cells following supplementation with IGF-1 for 14 days in either co-culture conditions (cCSC3C+Myo; cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. without (-) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using t test.

Figure Legend Snippet: A–C . Representative confocal microscopy images showing GFP pos (green) GATA-4 high c-kit pos cCSCs (cCSC4A+Myo) differentiated into the cardiomyocyte lineage (α-sarcomeric actin pos ; red) when co-cultured with adult rat cardiomyocytes (α-sarcomeric actin pos ; red) over 14 days (B). Note the lack of cardiomyogenic differentiation of GFP pos (green) GATA-4 high c-kit pos cCSCs at 3 days (cCSC4A+Myo; A) and GFP pos (green) GATA-4 low c-kit pos cCSCs at 14 days (cCSC3C+Myo; C). Bar = 20 µm. D . The percent number of GFP pos α-sarcomeric actin pos cells at 3, 7 and 14 days in vitro (DIV) following co-culture of GFP pos GATA-4 low expressing c-kit pos cCSCs with cardiomyocytes (cCSC3C+Myo), GFP pos GATA-4 high c-kit pos cCSCs with cardiomyocytes (cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. cCSC3C+Myo. ** vs. cCSC3C alone. † vs. cCSC4A alone. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. E . The percent number of GFP pos α-sarcomeric actin pos cCSC-derived cells following supplementation with IGF-1 for 14 days in either co-culture conditions (cCSC3C+Myo; cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. without (-) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using t test.

Techniques Used: Confocal Microscopy, Cell Culture, In Vitro, Co-Culture Assay, Expressing, Derivative Assay

A . ELISA assay assessed IGF-1 concentration in the culture medium, with and without FBS, of c-kit pos GATA-4 low cCSC1A, cCSC3C and c-kit pos GATA-4 high cCSC4A, when cultured alone. *P<0.05 vs. cCSC1A, †P<0.05 vs. cCSC3C. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . RT-PCR analysis showed c-kit pos GATA-4 high cCSC4A had low IGF-1 message levels, compared to rat fibroblasts (Fibro), skeletal muscle cells (SkM) and cardiomyocytes (CaM). C . RT-PCR analysis showed that IGF-1 gene expression level in fibroblasts (Fibro) and c-kit pos GATA-4 high cCSC4A (cCSC4A) is not altered through 7 days of culture in conditioned medium from adult cardiomyocytes (CM (CaM)). Also, IGF-1 gene expression level in cultured cardiomyocytes (CaM) is not altered through culture for 7 days with c-kit pos GATA-4 high cCSC4A conditioned medium (CM (cCSC4A)). D–E . c-kit pos GATA-4 high cCSC4A/cardiomyocyte co-culture conditioned medium, which is high in IGF-1 concentration, increased the percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (-) cCSC4A co-culture conditioned medium. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. F–H . Supplementation of IGF-1 to the culture medium attenuated percent number of TdT-positive cardiomyocytes, increased percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (−) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA.

Figure Legend Snippet: A . ELISA assay assessed IGF-1 concentration in the culture medium, with and without FBS, of c-kit pos GATA-4 low cCSC1A, cCSC3C and c-kit pos GATA-4 high cCSC4A, when cultured alone. *P<0.05 vs. cCSC1A, †P<0.05 vs. cCSC3C. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . RT-PCR analysis showed c-kit pos GATA-4 high cCSC4A had low IGF-1 message levels, compared to rat fibroblasts (Fibro), skeletal muscle cells (SkM) and cardiomyocytes (CaM). C . RT-PCR analysis showed that IGF-1 gene expression level in fibroblasts (Fibro) and c-kit pos GATA-4 high cCSC4A (cCSC4A) is not altered through 7 days of culture in conditioned medium from adult cardiomyocytes (CM (CaM)). Also, IGF-1 gene expression level in cultured cardiomyocytes (CaM) is not altered through culture for 7 days with c-kit pos GATA-4 high cCSC4A conditioned medium (CM (cCSC4A)). D–E . c-kit pos GATA-4 high cCSC4A/cardiomyocyte co-culture conditioned medium, which is high in IGF-1 concentration, increased the percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (-) cCSC4A co-culture conditioned medium. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. F–H . Supplementation of IGF-1 to the culture medium attenuated percent number of TdT-positive cardiomyocytes, increased percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (−) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA.

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Culture Assay

A. Representative Western blots show activation of the IGF-1 receptor (IGF-1-R) and downstream signaling to its physiological target, Akt, in cardiomyocytes co-cultured using inserts with c-kit pos GATA-4 high cCSC4A (cCSC4A+Myo), but not in cardiomyocytes cultured alone (Myo Alone) or when co-cultured with c-kit pos GATA-4 low cCSCs (cCSC3C+Myo) for 21 days. Addition of an IGF-1 receptor blocking antibody to the co-culture of c-kit pos GATA-4 high cCSC4A plus cardiomyocytes obliterates IGF-1 signalling in the co-cultured cardiomyocytes. B–C . Optical Density (O.D.) of phospho-IGF-1R (B) and phospho-Akt (C). *P<0.05 vs. cCSC4A+Myo. †P<0.05 vs. cCSC3C+Myo in B. †P<0.05 vs. cCSC3C+Myo, cCSC4A+Myo+IGF-1R Ab in C. Data are Mean ± SD of 3 assays and analysed using ANOVA. D . Representative Western blots shows prevention of Akt phosphorylation in cardiomyocytes when the Akt inhibitor, 124005 was added to the cardiomyocyte/c-kit pos GATA-4 high cCSC4A co-culture. E–F . Number of attached and percent number of beating cardiomyocytes decreased following inhibition of the IGF-1 signaling pathway through treatment with IGF-1 receptor blocking antibody or the Akt inhibitor, at day 19 for 48 hours. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. *P<0.05 vs. cCSC4A+Myo.

Figure Legend Snippet: A. Representative Western blots show activation of the IGF-1 receptor (IGF-1-R) and downstream signaling to its physiological target, Akt, in cardiomyocytes co-cultured using inserts with c-kit pos GATA-4 high cCSC4A (cCSC4A+Myo), but not in cardiomyocytes cultured alone (Myo Alone) or when co-cultured with c-kit pos GATA-4 low cCSCs (cCSC3C+Myo) for 21 days. Addition of an IGF-1 receptor blocking antibody to the co-culture of c-kit pos GATA-4 high cCSC4A plus cardiomyocytes obliterates IGF-1 signalling in the co-cultured cardiomyocytes. B–C . Optical Density (O.D.) of phospho-IGF-1R (B) and phospho-Akt (C). *P<0.05 vs. cCSC4A+Myo. †P<0.05 vs. cCSC3C+Myo in B. †P<0.05 vs. cCSC3C+Myo, cCSC4A+Myo+IGF-1R Ab in C. Data are Mean ± SD of 3 assays and analysed using ANOVA. D . Representative Western blots shows prevention of Akt phosphorylation in cardiomyocytes when the Akt inhibitor, 124005 was added to the cardiomyocyte/c-kit pos GATA-4 high cCSC4A co-culture. E–F . Number of attached and percent number of beating cardiomyocytes decreased following inhibition of the IGF-1 signaling pathway through treatment with IGF-1 receptor blocking antibody or the Akt inhibitor, at day 19 for 48 hours. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. *P<0.05 vs. cCSC4A+Myo.

Techniques Used: Western Blot, Activation Assay, Cell Culture, Blocking Assay, Co-Culture Assay, Inhibition

Blocking Assay:

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling
Article Snippet: Inc.) and VEGF, (RayBiotech, Inc.), according to the manufacturer's instruction. .. To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 μg/ml; Abcam) or Akt inhibitor (124005, 10 μmol/l; CalBiochem) added to the culture medium. ..

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Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A . VEGF concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, Fibro+Myo, cCSC4A+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . TGF-β1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. all. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. C . IGF-1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, †P<0.05 vs. Fibro+Myo, cCSC1A+Myo, cCSC3C+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. D. IGF-1 concentration correlated with cardiomyocyte contractility. Correlation was determined using Pearson Product Moment Correlation coefficient.

Article Snippet: To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 µg/ml; Abcam) or Akt inhibitor (124005, 10 µmol/l; CalBiochem) added to the culture medium.

Techniques: Concentration Assay, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A–C . Representative confocal microscopy images showing GFP pos (green) GATA-4 high c-kit pos cCSCs (cCSC4A+Myo) differentiated into the cardiomyocyte lineage (α-sarcomeric actin pos ; red) when co-cultured with adult rat cardiomyocytes (α-sarcomeric actin pos ; red) over 14 days (B). Note the lack of cardiomyogenic differentiation of GFP pos (green) GATA-4 high c-kit pos cCSCs at 3 days (cCSC4A+Myo; A) and GFP pos (green) GATA-4 low c-kit pos cCSCs at 14 days (cCSC3C+Myo; C). Bar = 20 µm. D . The percent number of GFP pos α-sarcomeric actin pos cells at 3, 7 and 14 days in vitro (DIV) following co-culture of GFP pos GATA-4 low expressing c-kit pos cCSCs with cardiomyocytes (cCSC3C+Myo), GFP pos GATA-4 high c-kit pos cCSCs with cardiomyocytes (cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. cCSC3C+Myo. ** vs. cCSC3C alone. † vs. cCSC4A alone. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. E . The percent number of GFP pos α-sarcomeric actin pos cCSC-derived cells following supplementation with IGF-1 for 14 days in either co-culture conditions (cCSC3C+Myo; cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. without (-) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using t test.

Techniques: Confocal Microscopy, Cell Culture, In Vitro, Co-Culture Assay, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A . ELISA assay assessed IGF-1 concentration in the culture medium, with and without FBS, of c-kit pos GATA-4 low cCSC1A, cCSC3C and c-kit pos GATA-4 high cCSC4A, when cultured alone. *P<0.05 vs. cCSC1A, †P<0.05 vs. cCSC3C. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . RT-PCR analysis showed c-kit pos GATA-4 high cCSC4A had low IGF-1 message levels, compared to rat fibroblasts (Fibro), skeletal muscle cells (SkM) and cardiomyocytes (CaM). C . RT-PCR analysis showed that IGF-1 gene expression level in fibroblasts (Fibro) and c-kit pos GATA-4 high cCSC4A (cCSC4A) is not altered through 7 days of culture in conditioned medium from adult cardiomyocytes (CM (CaM)). Also, IGF-1 gene expression level in cultured cardiomyocytes (CaM) is not altered through culture for 7 days with c-kit pos GATA-4 high cCSC4A conditioned medium (CM (cCSC4A)). D–E . c-kit pos GATA-4 high cCSC4A/cardiomyocyte co-culture conditioned medium, which is high in IGF-1 concentration, increased the percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (-) cCSC4A co-culture conditioned medium. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. F–H . Supplementation of IGF-1 to the culture medium attenuated percent number of TdT-positive cardiomyocytes, increased percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (−) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Culture Assay

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A. Representative Western blots show activation of the IGF-1 receptor (IGF-1-R) and downstream signaling to its physiological target, Akt, in cardiomyocytes co-cultured using inserts with c-kit pos GATA-4 high cCSC4A (cCSC4A+Myo), but not in cardiomyocytes cultured alone (Myo Alone) or when co-cultured with c-kit pos GATA-4 low cCSCs (cCSC3C+Myo) for 21 days. Addition of an IGF-1 receptor blocking antibody to the co-culture of c-kit pos GATA-4 high cCSC4A plus cardiomyocytes obliterates IGF-1 signalling in the co-cultured cardiomyocytes. B–C . Optical Density (O.D.) of phospho-IGF-1R (B) and phospho-Akt (C). *P<0.05 vs. cCSC4A+Myo. †P<0.05 vs. cCSC3C+Myo in B. †P<0.05 vs. cCSC3C+Myo, cCSC4A+Myo+IGF-1R Ab in C. Data are Mean ± SD of 3 assays and analysed using ANOVA. D . Representative Western blots shows prevention of Akt phosphorylation in cardiomyocytes when the Akt inhibitor, 124005 was added to the cardiomyocyte/c-kit pos GATA-4 high cCSC4A co-culture. E–F . Number of attached and percent number of beating cardiomyocytes decreased following inhibition of the IGF-1 signaling pathway through treatment with IGF-1 receptor blocking antibody or the Akt inhibitor, at day 19 for 48 hours. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. *P<0.05 vs. cCSC4A+Myo.

Techniques: Western Blot, Activation Assay, Cell Culture, Blocking Assay, Co-Culture Assay, Inhibition

Decreased αSMA promoter activity and αSMA protein expression in bleomycin-injured mice treated with IGF-1 receptor blocking antibody (A12). (A) Representative H&E sections of αSMA-GFP mice at day 21 after bleomycin injury. Fibrotic regions of lung parenchyma are indicated by ( ) and normal lung parenchyma are indicated by ( n ). (B) αSMA immunostaining of bleomycin-injured lung in an αSMA-GFP mouse. Note the overlap of αSMA staining (red) with αSMA-GFP expression (green) in the peribronchiolar fibrotic region indicated by ( ). Scale bars represent 100 μm. (C) (Left) Representative fluorescent images of αSMA-GFP mice treated with A12 (b and d) showed less αSMA promoter activity as indicated by GFP (green) positive cells, compared to control mice (a and c) at d21 after bleomycin instillation. (Right) Percentage of αSMA-GFP + cells/total number of DAPI + cells, quantification by NIH ImageJ (n = 4 mice/group, mean ± SEM). (D) (Left) Representative images of αSMA staining by immunofluorescent microscopy of the same A12-treated mice (b and d) compared to control mice (a and c) at d21 after bleomycin instillation. Large airways and vasculature staining for αSMA, indicated by an asterisk (*), were masked in the analysis. Interstitial staining, indicated by an arrow ( ), was included in the analysis. (Right) Ratio of αSMA staining area per DAPI + area (n = 4 mice/group, mean ± SEM).

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Decreased αSMA promoter activity and αSMA protein expression in bleomycin-injured mice treated with IGF-1 receptor blocking antibody (A12). (A) Representative H&E sections of αSMA-GFP mice at day 21 after bleomycin injury. Fibrotic regions of lung parenchyma are indicated by ( ) and normal lung parenchyma are indicated by ( n ). (B) αSMA immunostaining of bleomycin-injured lung in an αSMA-GFP mouse. Note the overlap of αSMA staining (red) with αSMA-GFP expression (green) in the peribronchiolar fibrotic region indicated by ( ). Scale bars represent 100 μm. (C) (Left) Representative fluorescent images of αSMA-GFP mice treated with A12 (b and d) showed less αSMA promoter activity as indicated by GFP (green) positive cells, compared to control mice (a and c) at d21 after bleomycin instillation. (Right) Percentage of αSMA-GFP + cells/total number of DAPI + cells, quantification by NIH ImageJ (n = 4 mice/group, mean ± SEM). (D) (Left) Representative images of αSMA staining by immunofluorescent microscopy of the same A12-treated mice (b and d) compared to control mice (a and c) at d21 after bleomycin instillation. Large airways and vasculature staining for αSMA, indicated by an asterisk (*), were masked in the analysis. Interstitial staining, indicated by an arrow ( ), was included in the analysis. (Right) Ratio of αSMA staining area per DAPI + area (n = 4 mice/group, mean ± SEM).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Activity Assay, Expressing, Blocking Assay, Immunostaining, Staining, Control, Microscopy

Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2 , Col1a1 , and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, * p < 0.05 compared to serum-free control).

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2 , Col1a1 , and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, * p < 0.05 compared to serum-free control).

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

IGF-1 treatment increases αSMA stress fibers. A . MLF treated with IGF-1 (100 ng/ml), TGF-β1 (10 ng/ml) or IGF-1/TGF-β1 (100 ng/ml and 10 ng/ml, respectively) with or without A12 (40 μg/ml) for 24 hr. Negative control is serum free media. Cells were co-stained for F-actin (red) and αSMA (green). B . The mean ratio (±SEM) of αSMA stress fiber (+) fibroblasts over all F-actin (+) fibroblasts is presented in the bar graph. * p < 0.05 compared to serum-free control. ** p < <0.01 compared to serum-free control.

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: IGF-1 treatment increases αSMA stress fibers. A . MLF treated with IGF-1 (100 ng/ml), TGF-β1 (10 ng/ml) or IGF-1/TGF-β1 (100 ng/ml and 10 ng/ml, respectively) with or without A12 (40 μg/ml) for 24 hr. Negative control is serum free media. Cells were co-stained for F-actin (red) and αSMA (green). B . The mean ratio (±SEM) of αSMA stress fiber (+) fibroblasts over all F-actin (+) fibroblasts is presented in the bar graph. * p < 0.05 compared to serum-free control. ** p < <0.01 compared to serum-free control.

Techniques: Negative Control, Staining, Control

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1) Product Images from "c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling"

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