Figure 3.
CASZ1 overexpression transforms Ba/F3 cells by activating the PI3K-AKT-mTOR signaling pathway. (A-B) Ba/F3 cells stably transduced with empty vector (Ba/F3-Ø) or CASZ1 (Ba/F3-CASZ1) were cultured in medium without growth factors. As a positive control, Ba/F3-Ø cells were also cultured in the presence of IL-3 (Ba/F3-Ø + IL-3). Viability (A) and proliferation (B) were determined at the indicated time points. Values represent the mean ± standard deviation of at least 3 independent experiments. **P<0.01; ***P<0.001 (C) Top 10 most significantly enriched KEGG pathways (adj. P<0.05) from upregulated genes (adj. P<0.05, log2 fold change>1) in the RNA-sequencing analysis of Ba/F3-CASZ1 versus Ba/F3-Ø cells. (D) Ba/F3-Ø and Ba/F3-CASZ1 cells were cultured in the indicated conditions for 24 hours. PI3K signaling activation was evaluated by immunoblot detection of the phosphorylation levels of AKT and S6. Total levels of CASZ1 and p27Kip1 were also analyzed. Lamin B was used as loading control. Data are representative of 2 independent experiments. (E) Ba/F3-CASZ1 cells were cultured for 24 hours in the presence of 100 or 500 nM of NVP-BEZ235 or vehicle (medium). PI3K signaling activation was evaluated by immunoblot detection of the phosphorylation levels of AKT. Total levels of CASZ1 and p27Kip1 were also analyzed. Lamin B was used as loading control. Data are representative of 2 independent experiments. (F, G) Ba/F3-CASZ1 cells were cultured in the presence or absence of 500 nM of NVP-BEZ235. Viability (F) and proliferation (G) were determined at the indicated time points. Values represent the mean ± standard deviation of experimental triplicates of a representative experiment (N=3). **P<0.01; ***P<0.001, one-way analysis of variance.