IR-A expression in endometrial carcinoma cell lines.

<p>A. Expression of IR-A (444 bp), IR–B (480 bp), IGF-2 (214 bp) and GAPDH (226 bp) mRNA in endometrial carcinoma cell lines was measured by RT-PCR. The results indicates that two transcript isoforms of insulin receptors (IR-A and IR–B) and IGF-2 were co-expressed in HEC-1-A, Ishikawa, KLE and RL95-2–CON cells. RL95-2–CON has a much higher ratio of IR–B expressed than IR-A. B. Real-time RT-PCR indicates the relative levels of IR-A and total IR mRNA in endometrial carcinoma cell lines, normalized to GAPDH. C.The ratio of IR-A/ IR in endometrial cancer cell lines from real-time RT-PCR results. D. ELISA quantitation of IGF-2 protein secreted by RL95-2–CON, RL95-2–IR-A, KLE, HEC-1-A and Ishikawa cells.</p

Effect of IR-A overexpression on downstream signaling pathways.

<p>A. Western blot analysis of IR protein expression and expression of downstream signaling proteins in RL95-2-CON, RL95-2–NC and RL95-2–IR-A cells. B. IR protein expression in RL95-2–IR-A cells is significantly higher than that in RL95-2–CON and RL95-2–NC cells. C. The relative expression of phospho-Akt is significantly increased and the relative expression of phosphorylated ERK1/2 reduced in RL95-2–IR-A cells than controls. *, #, <i>P</i> < 0.05 vs. control.</p

Expression of IR-A/ IR–B in endometrial carcinoma tissues and control normal endometrium tissues.

<p>A. Representative examples of the endometrial carcinoma tissues (total 103cases). Lane M shown the DNA marker and Lanes T1–T19 correspond to 19 separate endometrial carcinoma patients B. Representative examples of control normal endometrium tissues(total 60 cases). Lanes N1–N25 correspond to 25 separate patients with normal endometrium.</p

Tumorigenicity of the RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells in a xenograft model.

<p>A. Photographs of the inoculated BALB/c nude mice five weeks after inoculation, showing the tumor size. B. RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells were injected groups of five mice and tumor volumes were measured using calipers every week after the inoculation. C. Five weeks after inoculation, tumors were excised, fixed, and stained by hematoxylin and eosin (H&E; 400× magnification). *, <i>P</i> < 0.05 vs. control.</p

IR-A overexpression increases cell proliferation rate.

<p>A. Proliferation of RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells was measured over a 7-day period (plate 2000 cells/well as starting cells) B. Proliferation of the three cells was measured after 72 hours (plate 2000, 5000, 10000, 20000 cells/well as starting cells). 6 replicate wells were included per sample and the data points were present as means±SD. *, <i>P</i> < 0.05 vs. control.</p

PI3K inhibition reverses the effects of IR-A overexpression.

<p>A. Western blot analysis of phospho-Akt and total Akt levels 48 h after treatment with 5, 10, 20 and 40 µM of LY294002. B. Growth curves of RL95-2–IR-A cells following treatment with 20 µM LY294002 and without. C. Flow cytometry analysis indicating the proportion of RL95-2–IR-A cells containing S phase DNA at 48 h after treatment with 20 µM LY294002. D. Flow cytometry analysis indicating the percentage of apoptotic cells in RL95-2–IR-A cells at 48 h after treatment with 20 µM LY294002. *, <i>P</i> < 0.05 vs. control.</p