PMC

Cells were stained with a mouse anti-human IR antibody, followed by a goat anti-mouse 2^nd antibody conjugated with APC. The level of IR was measured by flow cytometry at channel FL4. A. LCC6 cells. B. T47D cells.

A. 1×10⁶ cells expressing luciferase were injected into the tail vein of athymic mice. Luciferase imaging of mice whole body were performed once a week. Representative images were shown at day 0, 14, 35 and 51. B. At day 51, after whole body imaging, mice were sacrificed and lungs and livers were removed and placed into 6 well plates. Ex vivo imaging was shown.

5×10⁶ LCC6 and IR shRNA clones were injected into the mammary fat pad. When tumors reached approximately 1000 mm³ in volume, mice were sacrificed and tumor metastases were examined in the lung sections. A. Representative images of lung micro-metastases were shown. The bar in each image represents 100 µm in length. B. One way ANOVA analysis was performed to compare the number of micro-metastasis in the lung of mice carrying LCC6 and the two shRNA clones. *, P<0.05; **, P<0.001.

A. LCC6, IR6 and IR14 cells were serum starved overnight, then treated with 20 nM of insulin for 15 minutes. Cellular lysates were blotted for phosphorylation of 46 kinase phosphorylation sites. Part A contains 28 antibodies printed in duplicate, and Part B contains 18 antibodies printed in duplicate. The arrows point at the phosphorylation of Akt Ser 473. B and C. LCC6, IR6 and IR14 cells (B) or T47D pRS and IR12 cells (C) were serum starved overnight, and then treated with or without 5 nM insulin, or 5 nM IGF-I for 10 minutes. Cellular lysates were separated by SDS-PAGE, and the phosphorylation levels of Akt at Ser473, ERK1/2 (MAPK) at Thr202/Tyr204, RSK at Ser380 and protein levels were assessed using specific antibodies by immunoblotting.

A. 5×10⁶ LCC6 and IR shRNA clones were injected into the mammary fat pad. Tumor growth was measured twice a week and is shown as tumor volume calculated using the formula (length × width × width/2). One way ANOVA analysis was performed to compare the tumor growth within the same day. *, P<0.0001. B. After 28 days of tumor growth, mice were sacrificed and tumor sections were stained for Ki67. Representative images were shown. The bar in each image represents 50 µm in length. C. One way ANOVA analysis was performed to compare the percentage of positive Ki67 staining between LCC6 and the two shRNA clones. *, P<0.05. D. Tumor sections were stained with TUNEL kit. Representative images were shown. The bar in each image represents 200 µm in length.

A. Real time RT-PCR analysis. LCC6 cells and IR shRNA clones were serum starved overnight, then treated with or without 20 nM of insulin for 24 hrs. Total RNA was isolated and gene expression profiles were analyzed using real-time RT-PCRs. mRNA levels were normalized to GAPDH mRNA levels. IR mRNA levels were used as a positive control for the RT-PCR analysis. One-way ANOVA analysis was done to compare the statistical significance among different cell lines. An unpaired t test was used to compare the difference between two treatment conditions. *, P<0.05; **, P<0.01. B. LCC6 cells and IR shRNA clones were incubated in different conditions as indicated for 48 hours. VEGF-A production in condition media was quantified by ELISA. One-way ANOVA analysis was done to compare the difference within the same treatment group. **, P<0.001.

A. T47D and IR12 clone were treated with or without insulin for 5 days. Cell numbers were estimated by MTT assay, and were shown as an absorbance at 570 nm. An unpaired t test was used to compare the difference between T47D and the shRNA clone. *, P<0.05. B. T47D cells, pRS vector cells and IR12 clone were mixed with 2% FBS in 0.45% agar and overlaid over 0.8% bottom agar. Colonies were imaged after 12 days of inoculation. Representative images were shown. C. The growth of LCC6 cells, pRS vector cells and two shRNA clones were measured by soft agar assay. Colonies formed were counted and averaged from 5 individual microscopic fields. The results are shown as the average number of colonies in 5 fields of three wells±S.E. The experiments were repeated three times with similar results. One way ANOVA analysis was performed to compare the difference between LCC6 and the two shRNA clones. *, P<0.05.

A. 5×10⁶ LCC6, pRS vector cells, and IR shRNA clones were injected into the mammary fat pad. Tumor growth was measured twice a week and is shown as tumor volume. Mice were sacrificed after 21 days of growth. One way ANOVA analysis was performed to compare the tumor growth within the same day. *, P<0.0001. B. Tumors were frozen in OCT solution. Tumor sections were stained with LYVE-1 antibody, followed by a Cy™2 conjugated secondary antibody and PE-conjugated CD31 antibody. Finally the sections were stained for the nucleus with DAPI. Confocal microscopy was performed and representative images of tumor periphery and tumor interiors were shown. The white dashed lines indicate the tumor boundary. Blue: DAPI; Green: LYVE-1; Red: CD31 (The yellow color is an overlay of red and green). The scale bar in the images is 50 µm.