Fig. 5

AMPK regulates EIF2A phosphorylation and is a pro-survival factor in Jurkat cells. a Jurkat cells were treated with or without FK866 at the indicated concentrations in the presence or absence of Compound C 5 μM for 48 h. Thereafter, cells were lysed and the levels of p-AMPK (Thr-172), p-MTOR, 4EBP1, p-4EBP1, BCL-2, MCL1, EIF2A and p-EIF2A were revealed by immunoblotting. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A. b In the same samples, caspase 3/7 activity was quantified and relative ATP levels were determined and then normalized to the number of viable cells (* indicates p-value <0.05). a and b are representative of a biological triplicate (mean and SD). c Jurkat cells were transduced with lentiviral particles containing scramble or shAMPK (targeting the α1 and the α2 subunit), then treated for 48 h with or without 5 nM FK866. Cell lysates were used for total AMPK, EIF2A, p-EIF2A, 4EBP1, p-4EBP1 and β-actin immunoblotting. WB analysis indicated 40 % of AMPK silencing (p-value < 0.05) and densitometric analysis shows the significant decrease of p-EIF2A in shAMPK Jurkat cells (p-value < 0.03). Mean and SD of a biological triplicate. d Jurkat scramble and shAMPK cells were treated for 48 h with the indicated doses of FK866. Cell viability was determined by PI staining and flow-cytometry (two biological replicates and statistics based on the acquisition of 10000 events/sample)