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. 2012 Nov 9;104(1):36–42. doi: 10.1111/cas.12032

Figure 4.

Figure 4

Inhibition of SCD1 expression decreases the nuclear localization and the transactivation potential of β‐catenin and impairs EMT‐like behavior. MCF7 and MDA‐MB‐231 were transfected with 10 nM of siRNA‐CTL or siRNA‐SCD1. After 48 h, the nuclear localization of β‐catenin was evaluated using cell fractionation and western blot analysis in MCF7 (a) and MDA‐MB‐231 (b) cells. Lap2 was used as a nuclear marker while α‐tubulin was used as a cytosolic marker. Upper graphs represent densitometry analysis of β‐catenin blots using Lap‐2 and α‐tubulin as nuclear and cytosolic loading controls, respectively. The bottom graphs represent densitometry quantification of SCD1 blots using α‐tubulin as a loading control. *P < 0.05. (c) The effect of SCD1 silencing was evaluated on the nuclear localization of β‐catenin. MCF7 cells were labeled with DAPI to stain the nuclei while β‐catenin and SCD1 were detected by immunofluorescence using specific antibodies. (d) MCF7 cells were co‐transfected with 10 nM of siRNA‐CTL (white bar) or siRNA‐SCD1 (black bars) mRNA and the TOP flash or the FOP flash (negative control) reporter constructs. After 48 h, cells were lysed and luciferase activity was determined.47 The results are expressed as a percentage of the ratio of TOP flash/FOP flash activity corrected by β‐galactosidase activity measured in cells transfected with the siRNA‐CTL. Results are representative of three independent experiments. *P < 0.05. Total RNA was extracted from MCF7 (e) and MDA‐MB‐231 cells (f). E‐cadherin and vimentin mRNA levels were evaluated by RT‐PCR. Hypoxanthine‐guanine phosphoribosyltransferase was used as a loading control. MCF7 (g) and MDA‐MB‐231 (h) cells were visualized and photographed under bright field illumination at ×20 magnification. MDA‐MB‐231 cells (i) were transfected and 48 h later cell invasion assays were performed. The graph compares the number of invasive siRNA‐CTL (white bar) and siRNA‐SCD1 (black bar) transfected cells. Results, representative of the three experiments, are expressed as a percentage of cells transfected with siRNA‐CTL. *P < 0.05.