Fig 2: Proposed model of PAK5-AIF signaling pathway in breast cancer proliferation. In breast cancer cells, on the one hand, PAK5 prevents AIF from releasing from mitochondria by changing mitochondrial membrane permeability and membrane potential; on the other hand, PAK5 prevents AIF entering the nucleus through the phosphorylation of AIF in cytoplasm, thus inhibiting cell apoptosis.

Fig 3: PAK5 inhibits AIF nuclear translocation by phosphorylation of AIF on Thr281. (a) Naturally occurring transcripts corresponding to the AIF precursor (b) AIF phosphorylation leads to decrease of nuclear translocation. Collection of cells for nuclear and cytoplasmic separation and western blot analysis. On the right side is the total protein extracted from the same batch of transfected cells, and the transfection efficiency of plasmids is detected by Western blot. (c) Analysis of phosphorylation sites and peptides of AIF by PAK5 by mass spectrometry. (d) PAK5 phosphorylated the T281 site of AIF. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (e) AIF T281A mutation can increase apoptosis. The apoptosis was detected by flow cytometry. The data are presented as a histogram of the mean _ SEM of three independent experiments (**p < 0.01, ***p < 0.001, n =3). The transfection efficiency of the plasmid was detected by Western blot (Fig.S8). (f) AIF T281A mutation and PAK5 interaction was weakened than AIF WT. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (g) AIF T281A mutation and importin α3 interaction was enhanced than AIF WT without affecting importinβ1. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (h)AIF phosphorylation promoted colony formation. Colonies of BT474 cells stably expressing control, AIF WT or AIF T281A were monitored. Representative pictures of the colonies are shown. The data are presented as a histogram of the mean _ SEM of three independent experiments (**p < 0.01, ***p < 0.001, n =3). (i) CCK8 was added at 0, 24, 36, 48, 72 hours of cell attachment in BT474 Cell, incubated at 37 ° for 2 hours, and detected by enzyme labeling instrument. The data are presented as a histogram of the mean _ SEM of three independent experiments (**p < 0.01, n =3). (j) BT474 cells (2× 106 cells) stably expressing vector control, AIF WT or AIF T281A were injected subcutaneously into the right flank of nude mice. Mice were imaged at 30 days after injection. Tumor diameter was measured at the indicated time points, and tumor volume was calculated. The results are presented as the mean SEM of 5 mice per group per time point (****p < 0.0001). (k) Tumors formed by cells described in (i) were extracted from mice and photographed. Tumor weight in mice from this experiment was measured upon autopsy at Day 30, and the results are presented as a histogram (**p < 0.01, ***p < 0.001).

Fig 4: PAK5 directly binds to AIF. (a,b) Endogenous AIF interacts with PAK5. BT474 cells/T47D lysates were immunoprecipitated with the anti-PAK5, anti-AIF or IgG. Precipitates were analyzed by western blot. (c) Exogenous PAK5 interacts with AIF. Total lysates were subjected to immunoprecipitation and western blot. (d) PAK5 directly binds to GST-AIF in vitro. Black stars indicate GST and GST-fusion proteins. (e) Co-localization of PAK5 and AIF. 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).

Fig 5: 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).