Effect of siUBE2E3 on SW480 membrane-associated E-cadherin.
SW480 cells were transfected with individual siRNA duplexes from the UBE2E3 SMARTpool for 72 hours: (A) Expression of ZEB1, E-cadherin and UBE2E3 were analysed by immunoblot. β-tubulin was used as a loading control. siRNA#1 (1*) shares the same 5’ nucleotide sequence as miR200 family seed sequence. The blot is representative of three individual experiments. Shown are cropped images, uncropped blots are included in S1 Raw images; (B) Quantitation of E-cadherin, ZEB1 and UBE2E3 protein levels upon siUBE2E3 knockdown in SW480 cells. Protein levels were determined using densitometry against the loading control β-tubulin and are representative for triplicate experiments (mean± SEM) *p<0.05 (p = 0.026 and p = 0.039 for si UBE2E3 1 and 3, respectively), **p<0.005 (p = 0.00247), ***p<0.001 for E-cadherin and UBE2E3 or duplicate experiments (mean ± sd) for ZEB1 *p = 0.023, **p = 0.0035, ***p<0.001; one-tailed unpaired t-test vs mock control; (C) Immunofluorescence staining of E-cadherin in fixed SW480 cells, 72 hours post treatment with siUBE2E3 siRNA duplexes #1, 2, 3 or mock control. Scale bar; 50μM.