Figure 5.

Dox-induced CD133 expression in BAKP (A,B) and POT (C) cells, as verified by immunoblot analysis, suppress caspase-3 mediated apoptotic cell death (D,F) and increases cell viability (E) after trametinib treatment (dose-response experiments). (A) Cells were incubated with Dox for the indicated times and subjected to immunoblot analysis with antibodies to CD133 and β-actin for normalization. (B) Immunoblot analyses with anti-CD133 of BAKP-CD133 and vector control BAKP-rtTA3 cells treated with increasing trametinib doses +/− Dox, as well as (C) POT-CD133 cells incubated +/− Dox for 72 h. (D) BAKP-CD133, BAKP-rtTA3 empty vector control, and POT-CD133 cells were exposed to increasing doses of trametinib for 72 h, and subjected to Annexin-FITC/PI apoptosis assays; apoptosis (%; D) and cell viability (%; E) of cells are shown. (F) Fluorescent FLICA Caspase 3/7 activity assays reveal a significant decline in caspase 3 activity in trametinib-treated CD133-expressing BAKP cells (+Dox), compared to uninduced cells (−Dox); representative merged images of red fluorescent FLICA- and GFP-positive cells (10×) (left panel); quantification of FLICA-positive cells (with active caspase-3, right panel). (G) Immunoblot analyses with antibodies to CD133, BCL-xL, and βActin of BAKP-CD133 treated with increasing trametinib doses +/− Dox. BAKP-CD133 cells were constitutively expressing GFP. Results shown are the means ± SD of three biological replicates of a representative experiment of three independent experiments. *, **, ***, **** represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Densitometric analysis comparing intensities of protein bands relative to bands with the highest intensity is shown in immunoblots.