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. 2017 Feb 21;116(7):912–922. doi: 10.1038/bjc.2017.39

Figure 6.

Figure 6

Cdk5 knockdown induces apoptosis in tumorspheres by increasing the pro-apoptotic protein Bim.(A) Cdk5 knockdown induces apoptosis in tumorspheres. Specific apoptosis in sphere-forming T24 cells is shown (mean±s.e.m., *P<0.01, n=3). (B) Cdk5 inhibition induces apoptosis in tumorspheres. Specific apoptosis of MCF7 tumorspheres after pretreatment of cells with roscovitine for 24 h before resuspension in fresh sphere-formation medium and cultivation for further 10 days in presence of roscovitine is shown (mean±s.e.m., *P<0.05, n=3). (C) The immunoblot shows levels of the pro-apoptotic protein Bim in non-targeting (nt) and Cdk5 shRNA cells. Equal loading is indicated (n=3). (D) Immunoblots show Bim protein in mitochondrial fractions of non-targeting (nt) and Cdk5 shRNA T24 cells at detachment of 3 h and 24 h. The mitochondrial marker COX IV indicates equal loading (n=3). (E) The bar graph displays Bim mRNA levels of non-targeting shRNA (nt) and Cdk5 shRNA cells (mean±s.e.m., *P<0.05, n=3). (F) Immunoblots indicate increased Foxo1 protein levels in Cdk5 knockdown T24 cells. Whole-protein bands indicate equal loading (n=3). (G) Immunoblots indicate increased Foxo1 protein levels in mesenchymal HMLE cells treated with roscovitine (24 h). Whole-protein bands indicate equal loading (n=3). (H) Cdk5 knockdown leads to an increase of Foxo1. Immunoblots show Foxo1 protein of cytosolic and nuclear fractionation of T24 cells. cAMP response element-binding protein (CREB) serves as marker for the nuclei fraction. Whole-protein bands indicate equal loading (n=3). (I) Immunostainings show Foxo1 protein (green) and nucleus (Hoechst33342, blue) of non-targeting (nt) and Cdk5 shRNA T24 cells (n=3).