PAK5 inhibits AIF release by regulating mitochondrial membrane permeability and potential. (a) PAK5 could increase the co-localization of AIF and COXIV (mitochondrial protein). Nucleus was stained with DAPI (4', 6 diamidino-2-phenylindole). Yellow indicates co-localization. Original magnification, × 600. The Pearson's correlation and overlap co-efficient were shown in bar graph format (30 cells) from three independent experiments were analyzed (error bars, SEM). (b,c) PAK5 could increase the expression of AIF in mitochondria. Cells were stably transfected with Flag-tagged PAK5 and extracted mitochondrial protein. Protein expression levels were determined by Western blotting. The data are shown as the mean _ SEM of triplicate experiments (*p < 0.05, **P < 0.01 vs. Control, n =3). (d-g) PAK5 could reduce membrane permeability of mitochondria. Cells were stably transfected with Flag-tagged PAK5 (d,e) or infected with PAK5-RNAi lentivirus (f,g) and extracted mitochondrial protein. Protein expression levels were determined by Western blotting. The data are shown as the mean _ SEM of triplicate experiments (***p < 0.001, ****p < 0.0001 vs. Control, n =3). (h-k) PAK5 can increase the membrane potential of mitochondria. Cells were stably transfected with Flag-tagged PAK5 (h,j) or infected with PAK5-RNAi lentivirus (i,k), the change in ΔΨm was examined using JC-1 staining assay. The ratio of fluorescent intensity of J-aggregates and monomers in treated cells is shown in e and f. The data are shown as the mean _ SEM of triplicate experiments (**P < 0.01, ***p < 0.001 vs. Control, n =3).