Effects of dansylcadaverine on cell proliferation.

<p>(A) MCF7, MCF10A, M12 and P69 cells were treated with dansylcadaverine, with or without IGF1, for 24 hr, after which absorbance was measured. (B) Stable MCF7/IGF1R KO and empty vector-transfected (control) cells were treated with dansylcadaverine for 24 hr, after which absorbance was measured. (*) significantly different versus control. Results of a representative experiment repeated three times with similar results are shown. The inset depicts the basal IGF1R levels in IGF1R-silenced and control (empty vector-transfected) cells. Heat shock protein-70 (hsp70) was used as a loading control).</p

Confocal microscopy analysis of IGF1R nuclear localization.

<p>Confocal immunofluorescence microscopic imaging of IGF1R-expressing MCF10A <b>(A, B)</b> and MCF7 <b>(D, E)</b> cells. Cells were transfected with an IGF1R siRNA or NT siRNA for 48 h. <b>Effect of IGF1 treatment on IGF1R expression</b>. Serum-starved MCF10A <b>(C)</b> and MCF7 <b>(F)</b> cells were treated with IGF1 (50 ng/ml) for 7 hr, and IGF1R localization was evaluated by fluorescence imaging. Control experiment using only secondary antibody <b>(G).</b> Fixed cells were stained for IGF1R with a fluorescent donkey anti-rabbit antibody (green- 488) and DAPI (blue). Results of a representative experiment repeated three times with similar results are shown. Images <b>A-G</b> were photographed using x63NA1.4 amplification.</p

Effects of dansylcadaverine and IGF1R inhibitors on cell proliferation of M12 cells.

<p>M12 cells were treated with dansylcadaverine, or with the selective IGF1R inhibitors AEW541 or AG1024, or both dansylcadaverine and IGF1R inhibitors for 24 hr, after which absorbance was measured. (*) significantly different versus control. Results of a representative experiment repeated three times with similar results are shown.</p

Effects of dansylcadaverine on IGF1R nuclear translocation in MCF7, MCF10A, M12 and P69 cells.

<p>Cells were treated with dansylcadaverine for 24 hr, after which cell fractionation was performed as described in Materials and Methods. <b>(A)</b> Western blot analysis of total IGF1R, total IR and tubulin (as a control for the cytoplasmic fraction) and lamin B1 (as a control for the nuclear fraction) in MCF7 and MCF10A cells. Given that different exposure times were carried out for the cytoplasmic and nuclear fractions, it is not possible to compare expression levels between both fractions. <b>(B)</b> Quantitative analysis of the results. Analyses were done using “ImageJ” software. <b>(C)</b> Western blot analysis of total IGF1R, tubulin and lamin B1 in M12 and P69 cells. <b>(D)</b> Quantitative analysis of the results. Results of a representative experiment repeated three times with similar results are shown. The “-“and “+” symbols represent control and dansylcadaverine-treated cells, respectively.</p

Confocal microscopy analysis of IGF1R nuclear localization in primary human fibroblasts.

<p>Fluorescence confocal microscope imaging of IGF1R in primary human fibroblasts. Cells were seeded in 24-well plates and after 24 hr were fixed and stained for IGF1R with a fluorescent donkey anti-rabbit antibody (green- 488) and DAPI (blue). Results of a representative experiment repeated two times with similar results are shown.</p

Western blot analysis of cytoplasmic and nuclear IGF1R in MCF10A cells.

<p>MCF10A cells were transfected with IGF1R siRNA (10 nM) or non-targeting (NT) siRNA and harvested 48 hr post transfection. (A) Western blot analysis of total IGF1R, Sumo-1, tubulin [a marker for the cytoplasmic <b>(C)</b> fraction] and SP1 [a marker for the nuclear <b>(N)</b> fraction]. (B) Densitometric analysis of Western blots. The results of a representative experiment repeated three times with similar results are shown. Analyses were done using the “ImageJ software”. Blots of the cytoplasmic fraction were exposed for short periods (2–5 min) whereas blots of the nuclear fraction were exposed for longer periods (10–15 min). Thus, it is not possible to compare between relative abundance of proteins between fractions.</p

Colony formation in soft agar.

<p>A total of 1.5×10⁴ Ishikawa cells and 1.2×10⁴ USPC-1 cells were seeded on 6-well plates in soft agar. The formed colonies were photographed at a magnification of 40× and 200× (A) and colonies with diameters greater than 3 mm were counted (B) at day 22. The bars represent the mean ± S.E.M. of three independent wells; * p<0.05 <i>versus</i> untreated cells.</p

Effect of vorinostat on IGF-I-mediated signal transduction.

<p>Ishikawa and USPC-2 cells were treated for 24 h with vorinostat (or left untreated) and/or IGF-I during the last 10 min of the incubation period. Whole-cell lysates (100 µg) were resolved on SDS-PAGE and immunoblotted with antibodies against pIGF-IR, TIGF-IR, pAKT, TAKT, pERK1/2, TERK1/2, p21, and actin. The figure shows the result of a typical experiment, repeated three times with similar results. IGF-IR phosphorylation in USPC-2 cells was significantly lower than in Ishikawa cells and was detected by Western blot analysis only after longer exposure times of blots to X-ray film. Therefore, this experiment does not allow comparison between both cell lines in terms of levels of expression or phosphorylation.</p

Regulation of IGF-IR promoter activity by vorinostat in endometrial cancer cells.

<p>Ishikawa and USPC-2 cells were transiently transfected with an IGF-IR promoter-luciferase reporter plasmid, p(−476/+640)LUC, which contains most of the proximal region of the IGF-IR promoter, and a ß-galactosidase vector. Promoter activity is expressed as luciferase values normalized for ß-galactosidase. A value of 100% was given to the promoter activity in the absence of vorinostat. Results are mean ± S.E.M. (three independent experiments); p*<0.01 <i>versus</i> untreated cells.</p

Effect of vorinostat on endometrial cancer cells proliferation.

<p>Cells were plated in 24-well plates at a density of 2×10⁴ cells/well for Ishikawa (A) and 3×10⁴ cells/well for USPC-2 (B). The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate; * p<0.05 <i>versus</i> untreated cells.</p