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: 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

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

doi: 10.1210/clinem/dgz057

Figure Lengend Snippet: The combination of FSH, GDF9, and BMP15 (GB) potentiate FSH-induced IGF2 expression in a dose-dependent manner. A. Primary human cumulus cells were treated for 48 hours with vehicle (C), GDF9 (G: 10 ng/mL), BMP15 (B: 10ng/mL), GDF9 and BMP15 combined (GB: 0.6, 2.5, and 10 ng/mL for each of G and B), in the presence or absence of follicle-stimulating hormone (FSH) (F: 50 ng/mL). IGF2 mRNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) and expressed relative to Rpl19. Columns represent the mean ± SEM (standard error of the mean). Columns with different letters differ significantly by one-way analysis of variance (ANOVA) analysis with Fisher’s least significant difference (LSD) test, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 9. Control and FSH groups were compared by t test; *P < 0.05, n = 9. B. Primary human cumulus cells were treated for 48 hours with vehicle (C), GDF9 (G: 5 ng/mL), BMP15 (B: 5 ng/mL), in the presence or absence of FSH (F: 5, 25, 100 ng/mL). IGF2 mRNA levels were determined by qPCR and expressed relative to Rpl19. Columns represent the mean ± SEM. Columns with different letters differ significantly by one-way ANOVA analysis with Fisher’s LSD test, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 5.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

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

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

doi: 10.1210/clinem/dgz057

Figure Lengend Snippet: The combination of GDF9 and BMP15 potentiates follicle-stimulating hormone (FSH)-induced IGF2 expression and protein synthesis. IGF2 protein levels were quantified by enzyme-linked immunosorbent assay. Columns represent the mean ± standard error of the mean, columns with different letters differ significantly by one-way analysis of variance analysis with repeated measures and Bonferroni correction a–b P < 0.001. One-way paired t-test was used to measure the difference between C and F, (**) for P < 0.01, n = 3.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

doi: 10.1210/clinem/dgz057

Figure Lengend Snippet: Effect of GDF9 and BMP15 (GB) on IGF2 promoter 3 activity. A. Cells were infected with lentivirus carrying the IGF2p3–LUC reporter and after overnight incubation treated for 48 hours with vehicle (C), follicle-stimulating hormone (FSH), FSH+GB, or GB. Luciferase activity was quantified and expressed relative to the control. Columns represent the mean ± SEM, columns with different letters differ significantly, n = 4. B. H19 transcripts were measured in cumulus cells treated with increasing doses of G+B and FSH (50 ng/mL) for 48 hours. Expression of H19 was determined by quantitative real-time polymerase chain reaction and expressed relative to Rpl19. Columns represent the mean ± standard error of the mean. Columns with different letters differ significantly by one-way analysis of variance analysis followed by Tukey’s multiple comparisons test (n = 5).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

Techniques: Activity Assay, Infection, Incubation, Luciferase, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

doi: 10.1210/clinem/dgz057

Figure Lengend Snippet: GDF9 and BMP15 regulate IGF2 via SMAD signaling. Primary human cumulus cells were treated for 48 hours with vehicle (C) or the combination of GDF9 and BMP15 (GB: 5 ng/mL) in the presence or absence of FSH (50 ng/mL), SMAD2/3 inhibitor SB431542 (SB: 1 µM), SMAD3 inhibitor SIS3 (SIS: 5 µM), or SMAD1/5/8 inhibitor LDN-193189 (LDN: 100 nM). IGF2 mRNA levels were determined by qPCR and expressed relative to Rpl19. t-Test was used to evaluate the difference between mRNA expression in the F+GB with and without the different SMAD inhibitors. Columns represent the mean ± standard error of the mean, columns with different letters differ significantly, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 8.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

Techniques: Expressing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

doi: 10.1210/clinem/dgz057

Figure Lengend Snippet: The combination of GDF9 and BMP15 potentiates FSH stimulation of IGF2 mRNA partially through IGF1R signaling. Primary human cumulus cells were treated for 48 hours, as described in Fig. 2A in the presence or absence of the IGF1R inhibitor NVP-AEW451 (1 μM). mRNA levels were determined by qPCR and expressed relative to Rpl19. One-way ANOVA analysis with repeated measures and Bonferroni correction indicated a global P = 0.008 for the group. Columns represent the mean ± SEM, columns with different letters differ significantly. A t-test revealed that the addition of NVP-AEW451 inhibits GDF9 and BMP15 potentiation of IGF2 mRNA (**P = 0.02), n = 4.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

Techniques:

Journal: Oncotarget

Article Title: IGFBP2 modulates the chemoresistant phenotype in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. Following 24-hour treatment with ON-TARGETplus IGFBP2 siRNA SMARTpool (si IGFBP2 pool), siNon-Targeting control (siNonT) or lipofectamine alone (Mock), Flo-1 cells were mock-treated or treated with 1 μg/mL (3.3 μM) CDDP, 2.5 μg/mL (8.3 μM) CDDP, 2.5 μg/mL (19.2 μM) 5-FU or 5 μg/mL (38.4 μM) 5-FU for 3 days and analyzed for cell viability using Cell Proliferation Reagent WST-1. Concurrent qRT-PCR was performed to verify IGFBP2 knockdown. B. Following 24-hour treatment with individual ON-TARGETplus IGFBP2 siRNAs, siNon-Targeting controls or lipofectamine alone, Flo-1 cells were mock-pretreated or pretreated with 200 ng/mL IGF1 or IGF2 for 1 hour followed by mock-treatment or treatment with 1 or 2.5 μg/mL (3.3 or 8.3 μM) CDDP in serum-free or 10% serum DMEM for 3 days. Cell viability was analyzed using Cell Proliferation Reagent WST-1. Columns and error bars are the mean ± SD of 3 or more wells in each experiment. Concurrent qRT-PCR was performed to verify IGFBP2 knockdown. (1 P, 1 μg/mL CDDP; 2.5 P, 2.5 μg/mL CDDP; 2.5 5-FU, 2.5 μg/mL 5-FU; 5 5-FU, 5 μg/mL 5-FU)

Article Snippet: Recombinant Human IGFBP2 (Cat#: 674-B2), IGF1 (Cat#: 291-G1), and IGF2 (Cat#: 292-G2) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Control, Quantitative RT-PCR, Knockdown