Fig 2: 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 ??m 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).

Fig 3: PAK5 inhibits apoptosis by blocking AIF nuclear translocation. (a) PAK5 directly binds to AIF amino acids 263 and 480. Black stars indicate GST and GST-fusion proteins. (b,c) Separation of nucleus and cytoplasm experiment showing that PAK5 expression decreased the nuclear translocation of AIF. The data are shown as the mean _ SEM of triplicate experiments (***p < 0.001, ****p < 0.0001 vs. Control, n =3). (d,e) PAK5 and AIF interact in the cytoplasm. Separation of cytoplasmic protein and nuclear protein with nucleoplasm separation Kit, lysates were subjected to immunoprecipitation and western blot with antibodies as indicated. (f) The interaction of AIF with importins was determined by immunoprecipitation assay. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (g) Overexpression of PAK5 diminished AIF interaction with importin a3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (h) Knockdown of PAK5 enhanced AIF interaction with importin a3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (i,j) Overexpression(i)/knockdown(j) of PAK5 had no effect on AIF interaction with importin a3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (k) PAK5 can inhibit apoptosis of breast cancer cells.BT474 cells transfected with the indicated constructs. The apoptosis was detected by flow cytometry. On the two-dimensional scatter diagram, PI and annexin V are x and Y axes respectively, the second quadrant represents apoptotic cells, and the fourth quadrant represents early apoptotic cells. The data are presented as a histogram of the mean _ SEM of three independent experiments (**p < 0.01, ***p < 0.001, n =3).

Fig 4: 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 5: PAK5 and AIF expression positively correlated with poor prognosis of breast cancer. (a) Analysis of expression differential proteins regulated by PAK5. Lysates from MDA-MB-231 cells overexpressing Flag-vector or Flag-tagged PAK5 and screening of differentially expressed proteins by sequencing. (b) Predict the expression of AIF in breast cancer and adjacent cancer in TCGA database. The fold-change was 0.897. (c) Predict the overall survival of AIF in GSE31519 database. (d) Predict the overall survival of AIF in TCGA database. (e) PAK5 and AIF expression in 122 clinical breast tissue pairs. Lysates of tumor tissues (T) and matched adjacent noncancerous tissues (N) were analyzed using Western blotting. Twelve representative pairs are shown. (f) The indicated protein levels in (e) were statistically analyzed (****p < 0.001). (g) Spearman's rank test was used to analyze the correlation between AIF relative expression and PAK5 relative expression in 122 subjects. (h) Representative images of immunohistochemical staining showing PAK5 and AIF protein expression in breast cancer. Original magnification, 400×. IHC staining was scored, and a Pearson correlation test was performed. Note that the scores of some samples overlap. (i-l) Kaplan-Meier survival analysis (GraphPad) of the relationship between overall survival (upper) and disease-free survival (lower) in breast cancer cases and PAK5 and/or AIF expression. The subjects were divided into different groups based on the indicated PAK5 and AIF expression scores in the tumors: PAK5 low (n = 35) and PAK5 high (n = 40, i); AIF low (n = 36) and AIF high (n = 39, j). n = 15 for PAK5 low/AIF low, n=4 for PAK5 high/AIF low, n=12 for PAK5 low/AIF high, n = 44 for PAK5 high/AIF high; n = 35 for high nuclear translocation ratio of AIF, n = 40 for low nuclear translocation ratio of AIF.