p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs

Nat. Cell Biol. 13, 317–323 (2011); published online 20 February 2011

ZEB1 is a protein of 1124 amino acids with a predicted relative molecular mass 124K. However, in Supplementary Fig. S5 (corresponding to Fig. 4a) of our manuscript, the uncropped image of the ZEB1 immunoblot shows two bands with a relative molecular mass of 200K and 125K, respectively. Two major ZEB1 signals (Mr 124K and 200K) have been observed in western blots when using different antibodies in various cell types (please see references 1,2,3,4 below). Therefore, to examine whether the bands observed in our study represent ZEB1, we knocked down ZEB1 in MCF12A control and p53R175H mutant cells using shRNA (Fig. 1a) and siRNA (Fig. 1b). As shown in the figure to the right, both of the bands are diminished by knockdown of ZEB1, indicating that these bands are ZEB1.

Figure 1: Downregulation of ZEB1 expression by shRNA or siRNAs in MCF12A control and p53R175H mutant cells.

(a) MCF12A cells expressing p53R175H mutant were infected with lentivirus encoding control shRNA or ZEB1 shRNA (clone ID: TRCN0000017566, Open Biosystems). After puromycine selection for 10 days, infected cells were subjected to immunoblotting analysis (ZEB1 (D80D3) antibody, #3396, Cell signalling; 1:1000). Immunoblot shows the expression of ZEB1 from MCF12A control and infected p53R175H cells. (b) MCF12A cells expressing p53R175H were transfected with two individual ZEB1 siRNAs (ZEB1-1, lane2, SASI_Hs02_00330526, Sigma; ZEB1-2, SASI_Hs02_00330527, Sigma). At 48-h post-transfection, transfected cells were subjected to immunoblotting analysis.

siRNA: SASI_Hs02_00330526 : GTTTGAAAAGATGCAAGCT; SASI_Hs02_00330527 : GTGGCCCATTACAGGCAAC shRNA: Hairpin sequence for TRCN0000017566


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