Figure 5.

IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE, #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.