Figure 5

shRNA targeting AKT1 enhances autophagy. (a) shRNA targeting human ATG4B (sh4B) reduces expression of a GFP-ATG4B fusion protein. 293ET cells expressing GFP-ATG4B (GFP-4B) with vector control or shRNA targeting ATG4B were analyzed for mean fluorescence intensity (MFI) by FACS 48 h after transfection. MFI is given as percentage of the control cell population (vector only). (b) sh4B reduces basal levels of dNGLUC release from cells expressing Actin-LC3-dN. 293ET cells were transfected with Actin-LC3-dN and sh4B or a control shRNA targeting GFP and released dNGLUC activity in the SN was detected after 48 h. Control cells express Actin-dN and shGFP. Error bars were calculated from three independent transfections (c) shRNA mediated knockdown of AKT1 induces dNGLUC release from Actin-LC3-dN. 293ET cells expressing Actin- LC3-dN with shATG4B, shAKT1 and vector control were cultured for 48 h prior to collection of SN and analysis of dNGLUC activity. (d) Generation of stable 293ET cells. 293ET cells were transduced with Actin-LC3-dN and selected at 0.3 μg/ml puromycin. Seventy-two hours after transfection with shRNA targeting GFP or AKT1, dNGLUC activity was determined in the supernatant from four independent trasnfections. RLU, relative light units. (Significances were calculated by a two-sided paired ttest as marked by asterisks: **, p < 0.01; ***, p < 0.001)