Journal: REPRODUCTION

Article Title: Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

doi: 10.1530/rep-10-0336

Figure Lengend Snippet: Figure 1 Effect of different IGF1 concentrations on the percentage of in vitro-produced blastocysts displaying different inner cell mass (ICM)/total cell number (TCN) proportions. Within ICM/TCN categories, bars with different letters differ significantly (P%0.05).

Article Snippet: For the IGF1 treatments, a vial containing 50 mg of lyophilised recombinant human IGF1 (291-G1, R&D systems, Wiesbaden-Norderstadt, Germany) was rehydrated with 500 ml of 0.1% PBS/BSA according to the manufacturer’s recommendations and stored at K20 8C in 10 ml aliquots (1000 ng).

Techniques: In Vitro, Produced

Journal: REPRODUCTION

Article Title: Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

doi: 10.1530/rep-10-0336

Figure Lengend Snippet: Figure 2 Relative transcript abundance (meanGS.E.M.) of develop- mentally important genes in day-8 blastocysts cultured in the presence of different IGF1 concentrations from the zygote stage onwards. Bars with different superscripts within each gene transcript indicate a significant difference (P%0.05).

Article Snippet: For the IGF1 treatments, a vial containing 50 mg of lyophilised recombinant human IGF1 (291-G1, R&D systems, Wiesbaden-Norderstadt, Germany) was rehydrated with 500 ml of 0.1% PBS/BSA according to the manufacturer’s recommendations and stored at K20 8C in 10 ml aliquots (1000 ng).

Techniques: Cell Culture

Journal: REPRODUCTION

Article Title: Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

doi: 10.1530/rep-10-0336

Figure Lengend Snippet: Figure 3 Three-dimensional reconstruction of confocal laser microscopy images showing protein localisation of the IGF1R and TP53 in day- 8 blastocysts cultured in the presence of different concentrations of IGF1. PI, propidium iodide. Note the lower expression of the IGF1R in the inner cell mass of the control group compared with the IGF1 groups and the lower expression of TP53 in the trophectoderm in the supraphysiolo- gical IGF1 group (1000 ng/ml).

Article Snippet: For the IGF1 treatments, a vial containing 50 mg of lyophilised recombinant human IGF1 (291-G1, R&D systems, Wiesbaden-Norderstadt, Germany) was rehydrated with 500 ml of 0.1% PBS/BSA according to the manufacturer’s recommendations and stored at K20 8C in 10 ml aliquots (1000 ng).

Techniques: Microscopy, Cell Culture, Expressing, Control

Journal: REPRODUCTION

Article Title: Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

doi: 10.1530/rep-10-0336

Figure Lengend Snippet: Figure 5 Relative signal strength (RSS) values (meanGS.E.M.) of TP53 in day-8 blastocysts treated with different concentrations of IGF1. ICM, inner cell mass; TE, trophectoderm. Between groups, bars with different superscripts indicate a significant difference (TE: a, b; total RSS: A, B; P%0.05). Within groups, the asterisk indicates significant differences between the ICM and the TE (P%0.05).

Article Snippet: For the IGF1 treatments, a vial containing 50 mg of lyophilised recombinant human IGF1 (291-G1, R&D systems, Wiesbaden-Norderstadt, Germany) was rehydrated with 500 ml of 0.1% PBS/BSA according to the manufacturer’s recommendations and stored at K20 8C in 10 ml aliquots (1000 ng).

Techniques:

Journal: REPRODUCTION

Article Title: Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

doi: 10.1530/rep-10-0336

Figure Lengend Snippet: Figure 4 Relative signal strength (RSS) values (meanGS.E.M.) of the IGF1R in day-8 blastocysts treated with different concentrations of IGF1 and displaying immunofluorescence in the inner cell mass (ICM) and the trophectoderm (TE). Between groups, bars with different super- scripts indicate a significant difference (ICM: a, b; TE: c, d; total RSS: A, B; P%0.05). Within groups, the asterisk indicates significant differences between the ICM and the TE (P%0.05).

Article Snippet: For the IGF1 treatments, a vial containing 50 mg of lyophilised recombinant human IGF1 (291-G1, R&D systems, Wiesbaden-Norderstadt, Germany) was rehydrated with 500 ml of 0.1% PBS/BSA according to the manufacturer’s recommendations and stored at K20 8C in 10 ml aliquots (1000 ng).

Techniques:

Journal: Journal of Molecular Endocrinology

Article Title: Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

doi: 10.1677/jme-09-0021

Figure Lengend Snippet: Figure 1 Expression of IR and IGF1R mRNA and proteins in HASMC. The relative amount of IGF1R to IR mRNA expression was measured by real-time RT-PCR, where IR was set to 1. The IGF1R and IR protein levels were analyzed by ELISA and are expressed as amount receptor protein relative to amount of total protein, IR was set to 1. The IGF1R to IR mRNA ratio (5.3G1.3) and the IGF1R to IR protein ratio (5.5G1.2) was found to be almost identical. ***Denotes P!0.001 (meanGS.E.M., nZ4).

Article Snippet: After washing away the unbound fraction with PBS–T, the secondary antibody was added and incubated on a shaker at RT for 2 h. The biotinylated anti-human IGF1R antibody (BAF 391; R&D Systems) was diluted as 0.5 mg/l in 0.1 w/v % HSA in PBS.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Endocrinology

Article Title: Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

doi: 10.1677/jme-09-0021

Figure Lengend Snippet: Figure 3 Detection of IR and IGF1R b-subunits in HASMC. IR and IGF1R b-subunit protein and evidence for the presence of hybrid IR/IGF1R were demonstrated by immunoprecipitation and western blot. Insulin receptor (IR) b-subunit (95 kDa) and IGF1R (IGF1R) b-subunit (97 kDa) were detected by western blot on the same membrane implying co-precipitation. Figures show blots after immunoprecipitation (IP) against either IR (left column, A and B), or IGF1R (right column, C and D) and immunoblot against the IR b-subunit (A and D) and the IGF1R b-subunit (B and C). Similar results were obtained in four independent experiments.

Article Snippet: After washing away the unbound fraction with PBS–T, the secondary antibody was added and incubated on a shaker at RT for 2 h. The biotinylated anti-human IGF1R antibody (BAF 391; R&D Systems) was diluted as 0.5 mg/l in 0.1 w/v % HSA in PBS.

Techniques: Immunoprecipitation, Western Blot, Membrane

Journal: Journal of Molecular Endocrinology

Article Title: Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

doi: 10.1677/jme-09-0021

Figure Lengend Snippet: Figure 4 (A) Activation of IR b-subunit in HASMC. Effect of insulin (dark gray bars), IGF1 (black bars), and IGF2 (gray bars) on tyrosine phosphorylation of IR b-subunit were analyzed by immunoprecipitation of IR by an anti-IR b-subunit antibody and immunoblotting with an anti-phosphotyrosine antibody (PY20) after exposure of near confluent HASMC to insulin, IGF1, and IGF2 at the concentrations of 10K10–10K8 mol/l for 10 min. Data are means from four independent experiments. Data were quantified by densitometry and are expressed as phosphor- ylated/total protein, fractional of maximal response. *P!0.05. (B) Activation of IGF1R b-subunit in HASMC. Effects of insulin (dark gray bars), IGF1 (black bars), and IGF2 (gray bars) on tyrosine phosphorylation of IGF1R b-subunit were analyzed by immunoprecipitation of IGF1R by an anti IGF1R b-subunit antibody and immunoblotting with an anti-phosphotyrosine antibody (PY20) after exposure of near confluent HASMC to insulin, IGF1, and IGF2 at the concentrations of 10K10– 10K8 mol/l for 10 min. Data are means from four independent experiments. Data were quantified by densitometry and are expressed as phosphorylated/total protein, fractional of maximal response. *P!0.05.

Article Snippet: After washing away the unbound fraction with PBS–T, the secondary antibody was added and incubated on a shaker at RT for 2 h. The biotinylated anti-human IGF1R antibody (BAF 391; R&D Systems) was diluted as 0.5 mg/l in 0.1 w/v % HSA in PBS.

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Western Blot

Journal: Journal of Molecular Endocrinology

Article Title: Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

doi: 10.1677/jme-09-0021

Figure Lengend Snippet: Figure 8 Proposed model for insulin resistance in HASMC. The insulin resistance of vascular smooth muscle is located at the receptor level. Due to the low number of IRs there are not enough receptors activated by insulin to generate a downstream signal. However, the IR b-subunit is activated by IGF1 when incorporated into hybrid IR/IGF1R. Both IGF1 and IGF2 activate the IGF1R and the hybrid IR/IGF1R and this elicits downstream signaling and biological effects.

Article Snippet: After washing away the unbound fraction with PBS–T, the secondary antibody was added and incubated on a shaker at RT for 2 h. The biotinylated anti-human IGF1R antibody (BAF 391; R&D Systems) was diluted as 0.5 mg/l in 0.1 w/v % HSA in PBS.

Techniques:

Journal: Nature Communications

Article Title: Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro

doi: 10.1038/s41467-021-24921-z

Figure Lengend Snippet: a Schematics timeline and protocol of ventricular-like CM differentiation from lateral plate mesoderm (LPM, Top), and its cardiac gene expression at day 30 (Bottom) from three independent differentiations. Data presented as mean ± SEM. Unpaired ttest with Welch’s correction was used for statistical comparison between groups. b Representative immunophenotypic characterization of ventricular-CM at day 30 from three independent differentiation. BMS = retinoid-x-receptor antagonist BMS189453. c Representative immunofluorescence images of Sarcomeric Actinin (Red) EdU + (green) ventricular CMs after co-culturing with PECs from three independent experiments. d Representative flow cytometric quantification of cTnT + Edu + ventricular CM in PEC co-culture after 6 days. The percentage in red was calculated by taking the cell count in Q2 and dividing by 100% cTnT + cells, or total events in Q1 and Q2 only (red box). Data presented in mean ± SEM ( n = 3). Two-tailed student T test was used. e Percentage of cTnT + EdU + VM in response to Linsitinib. Data presented in mean ± SEM ( n = 3 independent experiments) versus 0 µM Linsitinib. One-way ANOVA with Dunnett post hoc test was used. f Dose-response examination of IGF2 on CM proliferation. Data presented in mean ± SEM ( n = 3 independent experiments). One-way ANOVA with Dunnett post hoc test was used. g ELISA analysis of IGF2 in conditioned medium 2 and 6 days after co-culturing with PECs. Data presented in mean ± SEM ( n = 3 independent experiments). One-way ANOVA with Dunnett post hoc test was used. **** p value < 0.0001 versus CM only. h The expression of IGF2 RNA in PECs in response to increasing CM number. Data presented in mean ± SEM ( n = 3 independent experiments). One-way ANOVA with Dunnett post hoc test was used. **** p value < 0.0001 versus no CMs. i IGF2 expression in PECs in CM co-culture in transwell with or without RARα/γ antagonist BMS-189453 (BMS). Data presented in mean ± SEM ( n = 3 independent experiments). One-way ANOVA with Dunnett post hoc test was used. **** p < 0.0001 versus PECs-CM co-culture without BMS.

Article Snippet: IGF2 protein were quantified using Human IGF-II/IGF2 Quantikine ELISA kit DG200(R&D Systems, Minneapolis, MN).

Techniques: Expressing, Comparison, Immunofluorescence, Co-Culture Assay, Cell Counting, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Human reproduction (Oxford, England)

Article Title: Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.

doi: 10.1093/humrep/deu309

Figure Lengend Snippet: Figure7 PretreatmentofmouseblastocystswithIGF1improvestheirattachmenttoIshikawacellsinvitroandisabolishedbyLY294002.(A-D)Blastocyst appearance after 24 h pretreatment in SS medium in the presence or absence of 10 ng/ml IGF1 and/or in the presence of LY294002. (E) Attachment of blastocysts to Ishikawa cells under SS conditions after pretreatment in the presence of serum (black bars), with or without 2 mg/ml IGF1R neutralizing antibody (IGF1R nAb) or in SS medium (white bars) in the presence or absence of 10, 100 or 1000 ng/ml IGF1 or with 10 ng/ml IGF1 in the presence of 2 mg/ml IGF1R nAb or 5 mM LY294002 (PI3 kinase inhibitor) for 24 h. Results are displayed as the percentage of blastocysts attached to the Ishikawa cells, pooled from at least three experiments. N values in parentheses represent the total number of blastocysts added to the Ishikawa cells, pooled from three to nine experiments. Chi-square analysis was used to compare pre-selected treatment groups. Lines between bars show compared groups with sig- nificance. * indicates P , 0.05, ** indicates P , 0.01 and *** indicates P , 0.001.

Article Snippet: To determine a direct effect of IGF1 through the IGF1R, an IGF1R neutralizing antibody (IGF1R nAb; AF-305-NA; R&D Systems) was reconstituted at 0.2 mg/ml in PBS and used at a final concentration of 2 mg/ml.

Techniques: