antibody against human igf2  (R&D Systems)

Journal: PLoS ONE

Article Title: Rapamycin-independent IGF2 expression in Tsc2 -null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

doi: 10.1371/journal.pone.0197105

Figure Lengend Snippet: (A) TSC2 levels in human TSC2-null LAM 621–102 cells (TSC2—) cells and TSC2 re-expressing 621–103 LAM (TSC2++) cells. (B) RNA-Seq results show increased IGF2 transcripts per kilobase million (TPM) in TSC2— cells. (C) Corresponding plot of mapped reads along the hg38 reference genome corresponding to IGF2 . (D) Verification that Tsc2 is not expressed in Tsc2 -/- MEFs. (E) RNA-Seq results show increased Igf2 TPMs in Tsc2 -/- vs. Tsc2 +/+ MEFs. (F) Corresponding plot of mapped reads along the mm10 reference genome corresponding to Igf2 .

Article Snippet: IHC analysis of formalin-fixed paraffin-embedded control or LAM lung tissue was performed using specific antibodies against human IGF2 (Biorbyt, Cambridge, UK), phospho-ribosomal protein S6 (pS6, Cell Signaling Technology, Inc., Beverly, MA), and SM a-actin (Sigma Chemical Co., St. Louis, MO).

Techniques: Expressing, RNA Sequencing

Journal: PLoS ONE

Article Title: Rapamycin-independent IGF2 expression in Tsc2 -null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

doi: 10.1371/journal.pone.0197105

Figure Lengend Snippet: Representative images of IHC analysis show IGF2 expression in (A) LAM lesion and (B) LAM cluster detected with specific antibodies. Non-immune IgG was used as a control (see ). Igf2 expression in Tsc2 -/- MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) Tsc2 -/- MEFs were transfected with 50nM Igf2 siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and p -values reflect a sample size of 3 replicates.

Article Snippet: IHC analysis of formalin-fixed paraffin-embedded control or LAM lung tissue was performed using specific antibodies against human IGF2 (Biorbyt, Cambridge, UK), phospho-ribosomal protein S6 (pS6, Cell Signaling Technology, Inc., Beverly, MA), and SM a-actin (Sigma Chemical Co., St. Louis, MO).

Techniques: Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Staining

Journal: PLoS ONE

Article Title: Rapamycin-independent IGF2 expression in Tsc2 -null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

doi: 10.1371/journal.pone.0197105

Figure Lengend Snippet: (A) Re-expression of TSC2 (TSC2++) in TSC2-null LAM 102 (TSC2—) cells decreased STAT3 expression and activation. (B) siRNA-induced knockdown of STAT3 decreased STAT3 levels in TSC2— cells. (C) RNA-Seq results show upregulated IGF2 transcripts per kilobase million (TPM) in TSC2— cells transfected with either NT siRNA (Control) or STAT3 siRNA (siSTAT3). (D) STAT3 binding sites in human IGF2 and mouse Igf2 promoter regions. STAT3 enrichment in specific promoter regions of (E) human IGF2 and (F) mouse Igf2 genes was detected by ChIP-qPCR. Treatment of Tsc2 -/- MEFs with Stat3 inhibitor S3I-201 (100 nM for 18 hr) decreased Igf2 protein (G) expression as measured via Western blot and (H) secretion as measured via ELISA. (I) siRNA-mediated Stat3 knockdown also decreased Igf2 protein expression in Tsc2 -/- MEFs. (J) siRNA-mediated Igf2 knockdown decreased Stat3 phosphorylation but not total Stat3.

Article Snippet: IHC analysis of formalin-fixed paraffin-embedded control or LAM lung tissue was performed using specific antibodies against human IGF2 (Biorbyt, Cambridge, UK), phospho-ribosomal protein S6 (pS6, Cell Signaling Technology, Inc., Beverly, MA), and SM a-actin (Sigma Chemical Co., St. Louis, MO).

Techniques: Expressing, Activation Assay, Knockdown, RNA Sequencing, Transfection, Control, Binding Assay, ChIP-qPCR, Western Blot, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: Rapamycin-independent IGF2 expression in Tsc2 -null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

doi: 10.1371/journal.pone.0197105

Figure Lengend Snippet: Tsc2 -/- and Tsc2 +/+ MEFs were grown to near confluence, serum deprived for 2 hr and treated with indicated concentrations of rapamycin for 24 hr, followed by western blot analysis with indicated antibodies. (A) Treatment with 10nM rapamycin for 24 hr did not decrease Igf2 protein expression, although this dose completely suppressed pS6. (B) Igf2, Stat3, and pStat3 protein expression levels were unaffected by rapamycin treatment over a range of concentrations, while it completely inhibited pS6 at 2nM and 20nM concentrations. (C) IGF2 protein levels did not change in TSC2— and TSC2++ cells that were serum deprived for 2 hr and treated with 20nM rapamycin for 24 hr, as measured by western blot analysis. (D) IGF2 protein levels did not change in primary human LAM cells (LAM 111, LAM 105, LAM116) that were serum deprived for 2 hr and treated with 10nM rapamycin for 16 hr, as measured by western blot analysis. Images are representative of western blot analysis performed at least in three separate experiments.

Article Snippet: IHC analysis of formalin-fixed paraffin-embedded control or LAM lung tissue was performed using specific antibodies against human IGF2 (Biorbyt, Cambridge, UK), phospho-ribosomal protein S6 (pS6, Cell Signaling Technology, Inc., Beverly, MA), and SM a-actin (Sigma Chemical Co., St. Louis, MO).

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: ( a ) Tumour xenografts established from KYSE150-CON-shCON, KYSE150-Id1-shCON or KYSE150-Id1-shIGF2 ESCC cells were immunostained for CD31 and analysed for microvessel density (female 6–8-week-old nude mice, n =3 per group; scale bar, 100 μm). ( b ) Human VEGF (left panel) and mouse VEGF (right panel) concentration in serum of mice bearing Id1-overexpressing, Id1-shIGF2 or control tumours (female 6–8-week-old nude mice, n =6 per group) was analysed using ELISA. ( c , d ) Expression of VEGF and α-SMA ( c ) and secretion of VEGF ( d ) in fibroblasts fed with conditioned medium (CM) from KYSE150-CON-shCON, KYSE150-Id1-shCON or KYSE150-Id1-shIGF2 cells were assayed using western blot and ELISA, respectively. ( e , f ) Expression of VEGF and α-SMA ( e ) and secretion of VEGF ( f ) in fibroblasts treated with recombinant human IGF2. ( g , h ) Chemotactic migration of fibroblasts in response to conditioned medium (scale bar, 100 μm) from indicated ESCC cells ( g ) and exogenous IGF2 ( h ).Three biological replicates were performed for in vitro assays. Data in bar charts are presented as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 by Student's t- test.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Recombinant, Migration, In Vitro

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: ( a ) Western blot showing expression of IGF2 in ESCC (T) and matched non-tumour specimens (N), as well as expression of VEGF and α-SMA in CAFs and matched NEF from 11 ESCC patients. ( b ) Graphs showing positive correlation between IGF2 expression in oesophageal tissue and expressions of VEGF (left panel) and α-SMA (right panel) in fibroblasts, respectively, in 11 pairs of ESCC and adjacent normal tissues. Correlation was assessed using Pearson's rank correlation coefficient. ( c ) Comparison of serum IGF2 (left panel) and VEGF (middle panel) levels between healthy individuals ( n =50) and ESCC patients ( n =100); the data were pooled and a positive correlation was found between serum VEGF and IGF2 levels ( n =150) (right panel) using unpaired t -test. ( d ) Gene expressions were further divided into high and low levels using median expression level as the cut-off point for survival analyses. Kaplan–Meier curves comparing survival outcome of ESCC patients ( n =100) with high and low serum IGF2 levels (left panel), high and low serum VEGF levels (middle panel), and IGF2 High /VEGF High and IGF2 Low /VEGF Low levels (right panel); statistical significance was calculated by log-rank test.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Comparison

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: ( a ) Base pairing between 3′UTR of VEGF and miR-127-5p and miR-29c, respectively. ( b ) Quantification of miR-127-5p and miR-29c expressions in IGF2-treated fibroblasts by TagMan miRNA assay. ( c ) Western blot analysis showing the expression of VEGF in the fibroblasts transfected with miR-127-5p and miR-29c plasmids, respectively. ( d ) VEGF expression was determined in the fibroblasts transfected with miR-29c or miR-CON in the presence or absence of IGF2. ( e ) Comparison of miR-29c expression level between CAFs and matched NEFs from 11 ESCC patients (left panel; paired t -test), and correlation with oesophageal tissue IGF2 expression (right panel; Pearson's rank correlation coefficient). ( f ) VEGF expression in the fibroblasts transfected with miR-29c mimic (left panel) or inhibitor (right panel). ( g ) Luciferase activity in fibroblasts co-transfected with miR-29c and wild-type or mutant VEGF 3'UTR luciferase reporter plasmids. ( h ) miR-29c abrogated the stimulatory effect of IGF2 on migration of fibroblasts (scale bar, 100 μm). Three biological replicates were performed for in vitro assays. Bars, s.d.; * P <0.05; ** P <0.01; *** P <0.001 by Student's t -test.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Transfection, Comparison, Luciferase, Activity Assay, Mutagenesis, Migration, In Vitro

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: ( a ) Quantification of pri-miR-29c level in IGF2-treated fibroblasts using TagMan pri-miRNA assay. Data were normalized to U6 expression. ( b , c ) Fibroblasts were transfected with p53 overexpression or knockdown plasmids, and then expression levels of miR-29c ( b ) and VEGF ( c ) were determined by TagMan miRNA assay and western blot, respectively. ( d ) Three putative p53 binding sites in the promoter of miR-29c were identified by in silico prediction, and the enrichment of p53 in miR-29c promoter region was determined by ChIP. ( e ) Diagram illustrating the site-specific mutations introduced in the reporter plasmid for miR-29c promoter (Pgl3-hsa-miR29c-pro-BS3-WT) (upper panel). Lower panel showed the luciferase activity in fibroblasts transfected with p53 and wild type (WT) or mutated (M) miR-29c promoter. ( f ) Tagman miRNA assay and western blot analysis showing the expression of miR-29c and VEGF in p53 null fibroblasts upon IGF2 treatment, respectively. ( g ) VEGF expression in fibroblasts transfected with p53 or vector control in the presence or absence of IGF2. ( h ) Schematic diagram illustrating how IGF2 can induce fibroblasts to secrete VEGF via the mediation of miR-29c in a p53-dependent manner. Three biological replicates were performed for in vitro assays. Bars, s.d.; * P <0.05; ** P <0.01; *** P <0.001 by Student's t -test.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Expressing, Transfection, Over Expression, Knockdown, Western Blot, Binding Assay, In Silico, Plasmid Preparation, Luciferase, Activity Assay, Control, In Vitro

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: ( a ) Illustration within the box shows the experimental scheme for bone marrow transplantation; lower panel shows representative fluorescence images of recipient control mice without (labelled ‘No') and with bone marrow transplantation (labelled ‘Yes'); the red intensity indicated strong GFP signals. ( b , c ) Quantification of VEGFR1 + cells in bone marrow ( b ) and lung ( c ) of mice (female 6–8-week-old nude mice, n =3 per group) from each experimental group by flow cytometry analysis. ( d ) Representative three-dimensional tomography image of mice showing accumulation of GFP-positive cells in the thoracic region and in the subcutaneous tumour indicated by red frames (left panel) and quantification of VEGFR1 + cells in the subcutaneous tumours (right panel). ( e ) Migrating ability of sorted VEGFR1 + and VEGFR1 − bone marrow cells compared by FBS-gradient induced cell migration assay (scale bar, 100 μm). ( f ) The migration ability of VEGFR1 + bone marrow cells in response to the attraction of conditioned medium from IGF2-induced fibroblasts in the presence or absence of VEGF antibody was compared (scale bar, 100 μm). ( g ) Outline of experimental scheme and the effect of VEGFR1 blockade on tumour growth (female 6–8-week-old nude mice, n =6 per group; scale bar, 1 cm). ( h ) Experimental scheme and bioluminescence imaging showing effect of VEGFR1 blockade on lung metastasis; sections of lung tissue (H & E stained) are shown in the bottom panel (female 6–8-week old nude mice, n =8 per group; scale bars, 200 μm and 100 μm for top and bottom rows of photomicrographs, respectively). Bars, s.d.; * P <0.05; ** P <0.01, *** P <0.001 by Student's t -test.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Transplantation Assay, Fluorescence, Control, Flow Cytometry, Tomography, Cell Migration Assay, Migration, Imaging, Staining

Journal: Nature Communications

Article Title: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

doi: 10.1038/ncomms14399

Figure Lengend Snippet: IGF2 secreted by Id1-expressing cancer cells activates fibroblasts to secrete VEGF which exerts paracrine effects in the tumour microenvironment, as well as instigates VEGFR1 + bone marrow cells in the tumour macroenvironment to facilitate distant metastasis.

Article Snippet: The neutralizing antibodies against human IGF2 (#AF-292-NA), VEGF (#AF-293-NA) and CXCR2 (#MAB331-100) were purchased from R&D Company (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Molecular cancer research : MCR

Article Title: Intrinsic Resistance to Cixutumumab is Conferred by Distinct Isoforms of the Insulin Receptor

doi: 10.1158/1541-7786.MCR-15-0279

Figure Lengend Snippet: Insulin-like growth factor II (IGF-II) mediates resistance to cixutumumab in tumor cells over-expressing IR-B. Clonogenic assay in A549 cells that overexpress IR-B (A) in the presence or absence of cixutumumab and/or anti-IGF-II mAb. Represented are mean values +/− SEM, expressed as percentage of colony formation relative to control treated cells. Statistical significance was determined by one-way ANOVA followed by Tukey’s posthoc analysis. Statistically significant difference vs control treated cells (a), cixutumumab-treated cells (b), anti-IGF-II treated cells (c). (B, C) Western blot analysis of signal transduction in A549-IR-B cells treated with IGF-II (25 nM), in the presence or absence of cixutumumab and linsitinib. Serum-starved cells were pretreated with cixutumumab (100 nM) or linsitinib (0.01, 0.1 or 1 uM) for 15 min and 2 h, respectively, followed by incubation with rhIGF-II (25 nM) for 10 minutes. Proteins (15–20 ug) extracted from cultured cells were size-fractionated by SDS-PAGE and immunoblotted with anti-phospho-IRβY1150/51/IGF-IRβY1135/36, anti-phospho-AktS473 and anti-phospho-p42/p44 MAPKT202/Y204 antibodies. Total level of proteins was demonstrated by immunoblotting with antibodies directed against total Akt and p42/p44 MAPK. (D) MCF-7 and A549 cells with or without IR-A or IRB overexpression were treated with increasing concentrations of linsitinib (0.00015-10 uM) for 72 h, and tumor cell viability was quantified by CellTiter-Glo assay. The results are expressed as % of inhibition of tumor cell viability.

Article Snippet: Antibodies The following antibodies were purchased from commercial sources as indicated: mouse monoclonal antibodies against Akt (#2920) and p42/p44 MAPK (Erk1/2) (#9107), rabbit monoclonal antibodies against phospho-Akt S473 (#4060) and phospho-IGF-IR beta Y1135/1136 / Insulin Receptor beta Y1150/1151 (#3024), rabbit polyclonal antibody against phospho-p42/p44 MAPK T202/Y204 (Erk1/2) (#9101) (Cell Signalling, Beverly, MA, USA); mouse monoclonal antibody against IGF-IR (#MS-641-P) and Insulin Receptor (#MS-632-P) (Thermo Fisher Scientific, Fremont, CA, USA); rabbit polyclonal Insulin Receptor (#sc-711) and (#sc-7953) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal antibody against IGF-II (MAB292) and goat F(ab') 2 anti-mouse IgG-phycoerythrin (#F0102B) (R&D Systems, Minneapolis, MN, USA); goat polyclonal anti-mouse IRDye 680 conjugated (#926–32220) and anti-rabbit IRDye 800 conjugated (#926–32211) (LI-COR Biosciences, Lincoln, Nebraska, USA).

Techniques: Expressing, Clonogenic Assay, Control, Western Blot, Transduction, Incubation, Cell Culture, SDS Page, Over Expression, Glo Assay, Inhibition