Figure 7. p21 knockdown in Met-1 cells inhibits TCF1 and Cyclin D1 expression, resulting in sphere suppression.

(A, B) Met-1 and Met-1/p21shRNA (sh) cell lines that were untreated or treated with Wnt3a for 18 hrs, were tested by Taqman qRT-PCR for expression of LEF1 and TCF4 mRNA, or for (C) Axin 2 or LRP6 mRNA expression. Differences in mRNA expression between Met-1 and Met-1/p21shRNA were determined by unpaired t-test and significance set at p<0.05. (D) Met-1 and Met-1/p21sh cells were untreated or treated with Wnt3a for 18 hrs and tested by Western blot for expression of LEF-1, TCF-1, or Actin. (E) Met-1 cells were treated with control shRNA, TCF1shRNA1, and TCF1shRNA2 and levels of TCF1 mRNA were tested by qRT-PCR. (F) Control Met-1 and Met- 1/TCF1shRNA1 cells were tested for mammosphere formation; the percent of spheres in triplicate experiments is shown as mean ± SEM, p<0.05. (G) Met-1 control cells were compared to Met-1/TCF1shRNA1 and Met-1/p21shRNA cells by Western blot for expression of Cyclin D1 relative to Actin. (H) Met-1 cells were treated with control siRNA or Cyclin D1 siRNA and the level of mRNA knockdown was tested by qRT-PCR. (I) Met-1 and Met-1/CyclinD1 siRNA cells were tested for sphere formation. Results are shown as mean percent of spheres ± SEM; p<0.05.