Figure 4. Inhibition of eEF-2 kinase blunts autophagy, mitigates inhibition of protein synthesis and hastens reduction of cellular ATP.

(A) MCF-7 cells were transfected with a non-targeting RNA or an eEF-2 kinase-targeted siRNA (100 nM) for 72 h, and then treated with DPBS for 1 h. eEF-2 kinase, phosphor-EF-2, and the autophagy marker, LC3-II, were detected by Western blot. (B) MCF-7 cells with or without silencing of eEF-2 kinase were treated with DPBS; at the indicated times, cells were harvested for protein synthesis assay. Results shown are the mean ± SD of quadruplicate determinations from one of three identical experiments; *p<0.05, t-test. (C) MCF-7 cells transfected with 50 nM of NT RNA or an eEF-2 kinase siRNA were seeded in 96-well tissue culture plates (1×10⁴ cells per well). Forty-eight h later, the cells were starved in DPBS for 1 h, 2 h and 4 h. Cells were collected at the end of starvation for ATP assay. Results shown are the mean ± SD of quadruplicate determinations from one of three identical experiments; *p<0.05, **p<0.01, t-test.