Fig 2: Effect of wild-type p53 transfection on INSR protein expression.(A) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with an INSR promoter luciferase reporter along with a wild-type p53 expression vector, as described in the legend to Figure 3. After 48 h cells were harvested and the expression of the endogenous INSR was assessed by Western blots. HSC70 was measured as a loading marker. Panels (B) and (C) show scanning densitometric analyses of INSR and p53 protein levels, respectively, normalized to HSC70. * p < 0.01 versus empty vector-transfected cells.

Fig 3: Effect of p53 on cell proliferation.IGF1R-KD, INSR-KD and control MCF7 cells were plated in 96-well plates in quadruplicate and, after 24 h, were transfected with p53-WT or empty vector (pCMV). Proliferation was measured after an additional 72 h using an XTT assay. A value of 100% was given to the cell number of control, untreated cells at the end of the incubation period. * p < 0.01 versus respective pCMV-transfected cells.

Fig 4: Effect of IGF1R and INSR inhibition on UGT2B15 gene expression. (A) MCF7 cells were treated with the selective IGF1R inhibitor AEW541 (10 mM) for 48 h (or left untreated, C), after which cells were harvested, total protein was prepared, and IGF1R, UGT2B15, and p53 levels were measured by Western blots. HSP70 levels were measured as a loading control. The bar graph denotes UGT2B15 and IGF1R levels in control (solid bars) and AEW541 treated cells (striped bars). (B) T47D cells were treated with the INSR inhibitor S961 (100 nM and 1 mM) for 2 h. Cells were then harvested and levels of phospho- and total-INSR, UGT2B15, and p53 levels were measured by Western blots. A value of 100% was given to control, untreated cells. * p < 0.01.

Fig 5: Expression of UGT2B15 in endometrial cancer cell lines. (A) Total RNA was obtained from confluent USPC-1 and USPC-2 endometrial cancer cell lines and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was assigned to the UGT2B15 mRNA levels in USPC-1 cells. * p < 0.01 vs. USPC-1 cells. (B) Total protein was obtained from confluent USPC-1 and USPC-2 cell lines and UGT2B15, IGF1R, INSR, and p53 protein levels were measured by Western blots. HSP70 levels were assessed as a loading control.