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