Journal: Endocrine Connections

Article Title: 3-M syndrome: a growth disorder associated with IGF2 silencing

doi: 10.1530/EC-13-0065

Figure Lengend Snippet: Primer sequences for methylation-specific PCR.

Article Snippet: IGF2 and H19 mRNA levels were assessed using TaqMan assays (Hs01005963_m1 and Hs00262142_g1) with a cyclophyllin A probe (4333763T) for control gene expression.

Techniques: Methylation

Journal: Endocrine Connections

Article Title: 3-M syndrome: a growth disorder associated with IGF2 silencing

doi: 10.1530/EC-13-0065

Figure Lengend Snippet: Primer sequences for pyrosequencing.

Article Snippet: IGF2 and H19 mRNA levels were assessed using TaqMan assays (Hs01005963_m1 and Hs00262142_g1) with a cyclophyllin A probe (4333763T) for control gene expression.

Techniques: Methylation, Sequencing

Journal: Endocrine Connections

Article Title: 3-M syndrome: a growth disorder associated with IGF2 silencing

doi: 10.1530/EC-13-0065

Figure Lengend Snippet: Top 20 up- and downregulated probesets in the 3-M group as a whole and in each cell line by mutation.

Article Snippet: IGF2 and H19 mRNA levels were assessed using TaqMan assays (Hs01005963_m1 and Hs00262142_g1) with a cyclophyllin A probe (4333763T) for control gene expression.

Techniques: Mutagenesis

Journal: Endocrine Connections

Article Title: 3-M syndrome: a growth disorder associated with IGF2 silencing

doi: 10.1530/EC-13-0065

Figure Lengend Snippet: Relative fold expression of (A) IGF2 and (B) H19 measured using quantitative PCR in all 3-M cell lines. Expression of IGF2 is reduced while H19 is increased. * P <0.05.

Article Snippet: IGF2 and H19 mRNA levels were assessed using TaqMan assays (Hs01005963_m1 and Hs00262142_g1) with a cyclophyllin A probe (4333763T) for control gene expression.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 4. IGF2 was experimentally demonstrated as a direct target of miR-615-5p in pancreatic cancer cells. (a) Putative miR-615-5p-binding sequences in IGF2 3′-UTR and a schematic diagram of the reporter constructs showing the IGF2 3′-UTR sequence (IGF2_WT) and the mutated IGF2 3′-UTR sequence (IGF2_MUT; the mutant nucleotides of the miR-615-5p-binding site are underlined). (b) The IGF2_WT reporter or the IGF2_MUT reporter was co-transfected with the Renilla luciferase reporter into BXPC-3 and SW1990 cells containing either the miR-615-5p mimic or the negative control. Luciferase activity was assayed 48 h after transfection. (c) The expression of IGF2 mRNA was measured by real-time PCR in BXPC-3 and SW1990 cells 48 h after transfection. Beta-actin was used as a housekeeping gene control. (d) IGF2 protein levels in BXPC-3 and SW1990 cell supernatants were detected by ELISA 48 h after transfection. (e) IGF2 protein in BXPC-3 and SW1990 cells was detected by western blot 48 h post transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Article Snippet: IGF2 levels in the BXPC-3 and SW1990 cell Oncogene (2015) 1629 – 1640 © 2015 Macmillan Publishers Limited supernatants were detected using a human IGF2 ELISA kit (Cusabio Biotech, Newark, DE, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay, Construct, Sequencing, Mutagenesis, Transfection, Luciferase, Negative Control, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 5. miR-615-5p suppressed pancreatic cancer cell proliferation by directly targeting IGF2. (a) Immunofluorescence. BXPC-3 and SW1990 cells were fixed and incubated with primary antibodies for IGF2 and then Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The fluorescence level of IGF2 was notably decreased in miR-615-5p-transfected cells. Magnification: × 400. (b) BXPC-3 and SW1990 cell proliferation was detected by the MTT assay at 0, 24, 48 and 72 h after co-transfection with different combinations of NC or MIMIC with GFP or IGF2. For NC+GFP vs MIMIC+GFP: *Po0.05; **Po0.01. For NC+GFP vs NC+IGF2: #Po0.05; ##Po0.01. For NC+IGF2 vs MIMIC+IGF2: △△Po0.01. (c) The IGF2 protein was detected by western blot at 48 h post-transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Article Snippet: IGF2 levels in the BXPC-3 and SW1990 cell Oncogene (2015) 1629 – 1640 © 2015 Macmillan Publishers Limited supernatants were detected using a human IGF2 ELISA kit (Cusabio Biotech, Newark, DE, USA) according to the manufacturer’s instructions.

Techniques: Incubation, Staining, Transfection, MTT Assay, Cotransfection, Western Blot, Control

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 6. Imprinting status and expression levels of IGF2. (a) Gel photo of ApaI-digested PCR products for IGF2 genotyping. The heterozygous samples had two bands, which were considered as informative. (b) Gel photo of ApaI-digested RT–PCR products for imprinting status analysis. The ratios between the two bands are listed. The samples with a ratio of o3:1 or >1:3 had LOI of IGF2. (c, e) Results of qRT–PCR analysis of IGF2 mRNA expression in the 32 pairs of tissues shown on a column chart and a scattergram. (d, f) qRT–PCR results of miR-615-5p expression levels in the 32 pairs of tissues shown on a column chart and a scattergram. (g) The inverse correlation between IGF2 and miR-615-5p expression levels was examined by Pearson’s correlation analysis (R = −0.456, P = 0.033).

Article Snippet: IGF2 levels in the BXPC-3 and SW1990 cell Oncogene (2015) 1629 – 1640 © 2015 Macmillan Publishers Limited supernatants were detected using a human IGF2 ELISA kit (Cusabio Biotech, Newark, DE, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 7. The overexpression of miR-615-5p inhibited tumorigenicity in vivo. (a) The relative expression of miR-615-5p in the SW1990-vector group and the SW1990-mir-615 group was analyzed by qRT–PCR. (b, c) Photographs of tumors derived from the SW1990-vector group and the SW1990-mir-615 group of nude mice. (d, e) The graph is representative of tumor growth 25 days after inoculation. Tumor volume and weight were calculated, and all data are shown as mean ± s.d. (f) The expression of IGF2 was measured by immunohistochemistry in the tissues extracted from the SW1990-vector and SW1990-mir-615 nude mice. Magnification: × 40. (g) The percentage of IGF2-positive cells was calculated.

Article Snippet: IGF2 levels in the BXPC-3 and SW1990 cell Oncogene (2015) 1629 – 1640 © 2015 Macmillan Publishers Limited supernatants were detected using a human IGF2 ELISA kit (Cusabio Biotech, Newark, DE, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Expressing, Plasmid Preparation, Quantitative RT-PCR, Derivative Assay, Immunohistochemistry

Journal: Stem Cell Research & Therapy

Article Title: Insulin-like growth factor-II overexpression accelerates parthenogenetic stem cell differentiation into cardiomyocytes and improves cardiac function after acute myocardial infarction in mice

doi: 10.1186/s13287-020-1575-4

Figure Lengend Snippet: IGF-II overexpression promotes cardiomyocyte maturation in vivo. ESC- and PSC-derived cardiomyocytes at 1 × 10 5 cells/recipient mouse were injected into the heart at 1 week after acute myocardial infarction (MI). The IGF-II and VEGF levels in the injection sites ( a ) and the serum ( b ) were measured by ELISA at 4 weeks post-transplant. Data are expressed as the mean ± SD. * p < 0.05 vs. any other group. n = 12. c Immunofluorescence staining was performed to observe the cardiomyocyte morphology and to detect α-actinin expression (red staining) in the injection sites at 4 weeks post-transplant. Blue staining, Hoechst 33342. Green staining, eGFP. Scale bars, 10 μm

Article Snippet: IGF-II levels in serum (Quantikine ELISA Kit, cat# MG200, R&D Systems, Minneapolis, MN, USA), heart tissue (DuoSet ELISA, cat# DY792, R&D Systems), vascular endothelial growth factor (VEGF) levels in serum (cat# ab100751; Abcam), and heart tissue (cat# ab209882; Abcam) were determined with ELISA kits following the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Derivative Assay, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Insulin-like growth factors and related proteins in plasma and cerebrospinal fluids of HIV-positive individuals

doi: 10.1186/s12974-015-0288-6

Figure Lengend Snippet: IGFs and related protein levels in the plasma and CSF

Article Snippet: IGF2 levels were also quantified by ELISA (ALPCO Diagnostics, NH: cat # 22-IGF-E30, sensitivity 20 pg/ml) using the core service of the Einstein Diabetes Research Center (Biomarker Analytic Research Core, Bronx, NY, USA).

Techniques: Clinical Proteomics

Journal: Journal of Neuroinflammation

Article Title: Insulin-like growth factors and related proteins in plasma and cerebrospinal fluids of HIV-positive individuals

doi: 10.1186/s12974-015-0288-6

Figure Lengend Snippet: Comparison of IGFs and related proteins in plasma and CSF in subjects with or without neurocognitive impairment

Article Snippet: IGF2 levels were also quantified by ELISA (ALPCO Diagnostics, NH: cat # 22-IGF-E30, sensitivity 20 pg/ml) using the core service of the Einstein Diabetes Research Center (Biomarker Analytic Research Core, Bronx, NY, USA).

Techniques: Comparison, Clinical Proteomics

Journal: Frontiers in Physiology

Article Title: Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

doi: 10.3389/fphys.2017.00477

Figure Lengend Snippet: Let-7b has an enhanced inhibitory effect on IGF2BP3 expression in dwarf myoblast than in normal myoblast. (A) Desmin immunostaining of primary myoblast. (B) Relative let-7b expression in normal and dwarf myoblasts after transfection of let-7b mimic or NC mimic. (C) Relative mRNA expression after transfection of let-7b mimic or NC mimic in myoblast isolated from normal chicken. (D) Relative mRNA expression after transfection of let-7b mimic or NC mimic in myoblast isolated from dwarf chicken. (E) Relative IGF2BP3 protein expression in normal and dwarf myoblast after transfection of let-7b mimic or NC mimic. (F) ELISA analyzes of IGF2 protein expression in normal and dwarf myoblast after transfection of let-7b mimic or NC mimic. (G) Relative mRNA expression after transfection of let-7b inhibitor or NC inhibitor in myoblast isolated from normal chicken. (H) Relative mRNA expression after transfection of let-7b inhibitor or NC inhibitor in myoblast isolated from dwarf chicken. (I) Relative IGF2BP3 protein expression in normal and dwarf myoblast after transfection of let-7b inhibitor or NC inhibitor. (J) Relative luciferase activity of dwarf and normal myoblasts transfected with let-7b mimics and pmirGLO-IGF2BP3-3′UTR. Data were displayed as normalized fold change in relative luciferase activity (Firefly luciferase/Renilla luciferase, relative value of NC group in normal myoblast was set as 1). The data in (B–D,F–H,J) are mean ± S.E.M. with three cultures per group, and three wells per culture were assayed ( n = 9/treatment group). The data in (E,I) are mean ± S.E.M. from three independent experiments done in duplicate ( n = 6/treatment group). For (B–I) , independent sample t -test was used to analyze the statistical differences between groups. * p < 0.05; ** p < 0.01. For (F,J) , different letters above the bars indicate significant differences ( p < 0.05) by using the Duncan's Multiple Range Test, at the p < 0.05 significance level.

Article Snippet: The levels of IGF2 were determined using chicken IGF2 ELISA kit (CUSABIO BIOTECH Co. Ltd. China), following the manufacturer's instructions.

Techniques: Expressing, Immunostaining, Transfection, Isolation, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

Journal: Frontiers in Physiology

Article Title: Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

doi: 10.3389/fphys.2017.00477

Figure Lengend Snippet: Schematic illustration for signaling pathways of skeletal muscle growth regulated by let-7b. let-7b can inhibit skeletal muscle development through let-7b-IGF2BP3-IGF2 signaling pathway and let-7b-GHR-GHR downstream genes pathway in normal chicken. However, the large deletion mutation at the exon 10 and 3′UTR of GHR gene in dwarf chicken lead to disruption of let-7b binding site in GHR 3′UTR. In this case, let-7b would enhance its inhibition on IGF2BP3 expression. Therefore, let-7b inhibits skeletal muscle development only through let-7b-IGF2BP3-IGF2 signaling pathway with enhancing effect in dwarf chicken.

Article Snippet: The levels of IGF2 were determined using chicken IGF2 ELISA kit (CUSABIO BIOTECH Co. Ltd. China), following the manufacturer's instructions.

Techniques: Protein-Protein interactions, Mutagenesis, Disruption, Binding Assay, Inhibition, Expressing

Journal: bioRxiv

Article Title: Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a healthy pregnancy

doi: 10.1101/2021.04.27.441617

Figure Lengend Snippet: Placentas were collected from gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Frozen placental sections underwent periodic acid-Schiff (PAS) staining. Glycogen is stained red to purple. (B) Frozen placental sections were subjected to PL-I and PL-II in situ hybridization staining using RNAscope 2.5 HD Assay-BROWN kit. The PL-I and PL-II mRNAs are stained dark brown. (C) Quantification of relative intensity of Western blotting signals from . Data are presented as the mean fold changes relative to GD15 Ascl1 fl/fl mice (± SD; n = 3 for each group). *, P < 0.05, between Ascl1 fl/fl and hep-Ascl1 -/- mice. AKT, total protein kinase B; Ascl1 , achaete-scute homolog 1; ERK1/2, extracellular signal-regulated kinase 1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IGF2, insulin-like growth factor; PL-II, placental lactogen II.

Article Snippet: Serum IGF2 levels were quantified using a 1/6 dilution with the Mouse IGF-2 ELISA Kit (Boster Biological Technology, EK0381) as per directions by the manufacturer and read with the SpectraMax M2e spectrophotometer using the SoftMax Pro 6 program.

Techniques: Staining, In Situ Hybridization, RNAscope, HD Assay, Western Blot

Journal: bioRxiv

Article Title: Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a healthy pregnancy

doi: 10.1101/2021.04.27.441617

Figure Lengend Snippet: Maternal livers were collected and weighed from nonpregnant (NP) and gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Hepatic Igf2 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (B) Western blotting was performed using liver lysates with an antibody against IGF2. (C) Igf2 in situ hybridization on liver sections. (D) IGF2 immunostaining. (E) Levels of hepatic Igf2 promoter-specific transcript variants were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (F) Levels of IGF2 protein in serum were measured using ELISA and presented as the mean fold changes ± SD (n = 3-5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. P0, placental-specific Igf2 promoter; P1-3, placental- and fetal liver-specific Igf2 promoter.

Article Snippet: Serum IGF2 levels were quantified using a 1/6 dilution with the Mouse IGF-2 ELISA Kit (Boster Biological Technology, EK0381) as per directions by the manufacturer and read with the SpectraMax M2e spectrophotometer using the SoftMax Pro 6 program.

Techniques: Quantitative RT-PCR, Western Blot, In Situ Hybridization, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Diabetes

Article Title: Uncoupling Exercise Bioenergetics From Systemic Metabolic Homeostasis by Conditional Inactivation of Baf60 in Skeletal Muscle.

doi: 10.2337/db17-0367

Figure Lengend Snippet: Figure 7—Baf60 deficiency enhances the expression and secretion of IGF2. A: Heat map representation of Baf60-regulated genes encoding secreted factors. B: qPCR analysis of gene expression in quadriceps muscle from AKO (n = 3), CKO (n = 6), ACKO (n = 4–5) mice and their littermate controls (Ctrl) on standard chow diet. C: IGF2 secretion in ex vivo cultured soleus (left) or plantaris (right) muscles. D: Plasma IGF2 concentration in chow-fed control and ACKO mice (n = 4–5). E: qPCR analysis of quadriceps gene expression in HFD-fed mice (left, n = 5–11) and plasma IGF2 concentrations in HFD-fed mice (right, n = 7–9). F: qPCR analysis of quadriceps gene expression (left) and plasma IGF2 levels (right) in old control and ACKO mice (n = 6–7). G: ChIP assays on control and ACKO muscle nuclei using antibodies against histone 3 acetylation (AcH3) and H3K9 demethylation (H3K9me2). Location of primers on the proximal regions of three IGF2 promoters is shown. H: Schematic model demonstrating the regulation of exercise preformation and systemic energy homeostasis by Baf60a and Baf60c. Data in B–G represent mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: Then, 200 mL reagent diluent was added to each sample to dissolve, from which 100 mL was used to measure insulin-like growth factor 2 (IGF2) levels using a Mouse IGF-II DuoSet ELISA Kit (R&D systems) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Ex Vivo, Cell Culture, Muscles, Clinical Proteomics, Concentration Assay, Control

Journal: BMC Genetics

Article Title: Common polymorphism in H19 associated with birthweight and cord blood IGF-II levels in humans

doi: 10.1186/1471-2156-6-22

Figure Lengend Snippet: Birth size and cord IGF-II levels by offspring H19 2992 genotype (ALSPAC cohort)

Article Snippet: Cord blood samples were collected at birth in 338 of these infants and assayed for IGF-II levels as previously reported [ ]; these infants had larger birthweight (mean = 3546 g) than infants for whom cord blood was unavailable (3467 g), but were no different in H19 +2992 genotype (chi-square: P = 0.7) The Cambridge (Wellbeing) birth cohort was studied to provide independent confirmation of mother's H19 genotype associations.

Techniques:

Journal: BMC Genetics

Article Title: Common polymorphism in H19 associated with birthweight and cord blood IGF-II levels in humans

doi: 10.1186/1471-2156-6-22

Figure Lengend Snippet: Birthweight and IGF-II levels by maternal H19 2992 genotype, within CC genotype offspring (i.e. effect of mother's untransmitted allele), (ALSPAC cohort)

Article Snippet: Cord blood samples were collected at birth in 338 of these infants and assayed for IGF-II levels as previously reported [ ]; these infants had larger birthweight (mean = 3546 g) than infants for whom cord blood was unavailable (3467 g), but were no different in H19 +2992 genotype (chi-square: P = 0.7) The Cambridge (Wellbeing) birth cohort was studied to provide independent confirmation of mother's H19 genotype associations.

Techniques:

Journal: BMC Genetics

Article Title: Common polymorphism in H19 associated with birthweight and cord blood IGF-II levels in humans

doi: 10.1186/1471-2156-6-22

Figure Lengend Snippet: Birthweight and IGF-II levels by offspring H19 2992 genotype, within CC genotype mother's (i.e. effect of fetal genotype), (ALSPAC cohort)

Article Snippet: Cord blood samples were collected at birth in 338 of these infants and assayed for IGF-II levels as previously reported [ ]; these infants had larger birthweight (mean = 3546 g) than infants for whom cord blood was unavailable (3467 g), but were no different in H19 +2992 genotype (chi-square: P = 0.7) The Cambridge (Wellbeing) birth cohort was studied to provide independent confirmation of mother's H19 genotype associations.

Techniques: