Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) QMS workflow to identify the PAK4 interactome. MS analysis was performed using MCF7 cells stably expressing FLAG-PAK4 or FLAG-BAP (control). WC lysate and Cyt and Nuc subcellular fractions were analyzed in four independent biological replicates. After IP with anti-FLAG antibody and elution from FLAG beads with FLAG peptides, all samples were digested with trypsin and each labeled with a different iTRAQ 8-plex reagent. The eight iTRAQ labeled samples (four replicates of each FLAG-PAK4 and FLAG-BAP) were pooled and subjected to nano-LC-MS/MS analysis, followed by statistical and bioinformatic analysis. (B) Verification of subcellular fractionation. Lysates from WC, Cyt and Nuc fractions of FLAG-PAK4 and FLAG-BAP stably transfected MCF7 cells were analyzed by immunoblotting. Vinculin was used as a cytoplasmic marker; pRb as nuclear marker. (C) Schematic of the number of proteins identified by QMS in the different fractions before and after cut-off. Top: Total number of proteins recognized by QMS in each cellular fraction; Middle: Number of proteins in each fraction after cut-off; Bottom: Total number of unique proteins in all the fractions after cut-off. The cut-off criteria for specific FLAG-PAK4 associated hits was a combination of 5% FDR and above the 99.9% confidence interval of FLAG-BAP. (D) Venn diagram showing the number of specific PAK4 interacting proteins in WC and subcellular fractions.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Stable Transfection, Expressing, Control, Labeling, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Fractionation, Transfection, Western Blot, Marker

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Whole cell lysates derived from MCF7 cells stably expressing FLAG-PAK4 or FLAG-BAP were used for validation of QMS hits. After anti-FLAG IP and elution with FLAG peptides, samples were subjected to immunoblot analysis for the indicated proteins. Anti-FLAG (4 th row) and anti-PAK4 (5 th row) antibodies were used as controls. The left input panel shows immunoblotting of the two lysates. (B) PAK4 interactome networks obtained from the STRING database with the clusters identified by AutoAnnotate and visualized by Cytoscape. Diamond nodes: PAK4 interactors identified in the whole cell, or in both cytoplasmic and nuclear fractions or in all three fractions; Circle nodes: interactors identified in the cytoplasmic fraction or in both whole cell and cytoplasmic fraction; Squared nodes: interactors identified in the nuclear fraction or in both whole cell and nuclear fraction; Gray nodes: previously described PAK4 interactors.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Derivative Assay, Stable Transfection, Expressing, Biomarker Discovery, Western Blot

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Several subunits of the Arp2/3 and CCT complexes were identified in the PAK4 interactome. White nodes: proteins passed QMS cut-off; Grey nodes: proteins appeared in MS but did not pass the QMS cut-off; Darker grey node: not in the MS list. (B) After GFP-Trap IP of H1299 cell lysates transiently expressing EGFP (control) or EGFP-PAK4, samples were subjected to immunoblot analysis for the indicated proteins. The upper panel is blotted with anti-CCTε, the middle panel with anti-ARPC2, while anti-GFP was used to control the IP efficiency in the lower panel. Input lanes are direct immunoblot of the used cell lysates. (C) After anti-CCTε IP of H1299 cell lysates transiently expressing EGFP-PAK4, blots were probed with an anti-GFP antibody in the upper panel. Anti-CCTε was used to control the IP efficiency in the lower panel. Input lane shows direct immunoblotting of the used lysate.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Expressing, Control, Western Blot

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) PAK4 mediated phosphorylation was analyzed by an in vitro kinase assay using recombinant HIS-PAK4 together with the Arp2/3 complex (left panel) or GST-VCA (right panel) as substrates, with GST as a negative control and GST-RAF1 (332–344) as a positive control (upper panels). The lower panels display the protein loading in the assays by Coomassie Brilliant Blue staining. (B) HIS-PAK4 phosphorylation of the WASP VCA domain was analyzed using an anti-N-WASP pSer484/Ser485 antibody after a kinase assay using recombinant HIS-PAK4 with GST-VCA as a substrate. GST serves as a negative control, while the anti-RAF1 pSer338 antibody was used as a positive control to detect GST-RAF1 phosphorylated by PAK4 (upper panel). The lower panel shows the loading of HIS-PAK4 protein and GST-fusion proteins used in the assay by silver staining. (C) HIS-PAK4 was pulled-down in the presence of GST-VCA or the Arp2/3 complex with Ni-NTA agarose and input (I), supernatant (S) and pellet (P) analyzed by silver staining. (D) IP of EGFP control or EGFP-PAK4 transiently expressed in H1299 cells analyzed by immunoblotting using an anti-N-WASP antibody (upper panel right two lanes). The left two lanes show immunoblotting of the input lysates. Anti-GFP was used to control the expression and IP efficiency in the lower panel. (E) N-WASP was immunoprecipitated with an anti-N-WASP antibody from lysates of H1299 cells transiently expressing EGFP-PAK4 and samples were analyzed by immunoblot using an anti-GFP antibody with the lysate input to the left (upper panel). Anti-N-WASP was used to control the expression and IP efficiency in the lower panel. (F) PAK4 was immunoprecipitated with an anti-PAK4 antibody from lysates of MCF7 cells, with rabbit IgG as a control, samples were analyzed by immunoblot using an anti-N-WASP antibody with the lysate input to the left (upper panel). Anti-PAK4 blotting was used to control IP efficiency in the lower panel. (G) N-WASP was immunoprecipitated with an anti-N-WASP antibody from lysates of H1299 cells, with rabbit IgG as a control, samples were analyzed by immunoblot using an anti-PAK4 antibody with the lysate input to the left (upper panel). Anti-N-WASP blotting was used to control IP efficiency in the lower panel. (H) PAK4, N-WASP and F-actin co-localized in the cell periphery after re-plating. FLAG-PAK4 was labeled with an anti-FLAG mab (Green), N-WASP with an anti-N-WASP antibody (Red), F-actin with SiR-actin (Purple) and Nuclei with Hoechst (Blue), Scale bar: 10 μm.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Negative Control, Positive Control, Staining, Silver Staining, Control, Western Blot, Expressing, Immunoprecipitation, Labeling

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Immunoblotting of lysates from control siRNA transfected cells and PAK4 knockdown (siPAK4) cells with anti-N-WASP and anti-N-WASP pSer484/Ser485 antibodies. PAK4 knockdown efficiency was detected with an anti-PAK4 antibody and vinculin was used as a loading control. (B) Actin polymerization reactions were performed with actin, Arp2/3 complex, GST–VCA and GST-PAK4 KD (kinase domain) in different combinations as indicated. The amount of polymerized actin over time is indicated by the increase in fluorescence intensity. (C) G-actin and F-actin were separated by centrifugation in H1299 cell lysates with or without PAK4 siRNA-mediated knockdown. Left panel: Cytochalasin D (Cyto D) treatment was used as a control to block actin polymerization. Immunoblot analysis with an anti-actin antibody shows the amount of G-actin (G) and F-actin (F) for each condition. Right panel: PAK4 knockdown efficiency was assessed by immunoblotting using vinculin as a loading control (top). In addition, the total amounts of actin were analyzed (bottom). (D) siRNA knockdown of PAK4 alters the cellular morphology and F-actin distribution in H1299 cells. Nuclei were stained with Hoechst (Blue) and F-actin with Phalloidin (Red). Scale bar: 10 μm.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Western Blot, Control, Transfection, Knockdown, Fluorescence, Centrifugation, Blocking Assay, Staining

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 1. Effects of PAK4 overexpression and knockdown on mesenchymal transition in glioma cells. (a) Western blot analysis of 4910 and U251 glioma cells transfected with EV and PAK4-FL for 48 h along with untreated controls and GAPDH served as a loading control. (b) Representative micrographs of morphological characteristics in EV- or PAK4-FL-treated glioma cells after 48 h transfection. Scale bars: 10 μm. (c) Representative immunoblots from three independent experiments using whole cell lysates of glioma cells transfected with EV or PAK4-FL to assess changes in EMT markers. (d) Immunoblot analysis of effects of PAK4 downregulation using PAK4shRNA when compared with untreated and SV controls in 4910 and U251 cells. (e) Phase contrast micrographs of morphological characteristics of 4910 and U251 cells after SV and PAK4sh treatments for 48 h. Scale bars: 10 μm. (f) Confocal microscopy showing E-cadherin (green) and N-cadherin (red) expression in control, SV- and PAK4sh-treated cells. DAPI was used for nuclear counterstaining. Scale bars: 10 μm.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Over Expression, Knockdown, Western Blot, Transfection, Control, Confocal Microscopy, Expressing

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 6. Role of PAK4 in the regulation of PPARγ-mediated Nox1 and EMT in glioma cells. (a) Cells were subjected to SV and PAK4sh for 24 h and subsequently treated with IR for an additional 24 h. At the end of the treatments, whole-cell lysates were subjected to western blotting with GAPDH as internal loading control. (b) Cells were treated with EV and PPARγ-FL for 24 h followed by treatment with PAK4sh for an additional 24 h. Western blotting was performed with whole cell lysates; representative blots from three independent experiments are shown. (c) 4910 cells were treated independently with SV-control or PAK4sh or GW9662 (10 μM) or IR (8 Gy) or with combinations of SV+IR, PAK4sh+IR and GW9662+IR for 48 h. Total ROS levels were estimated as described in Materials and methods section and are presented as mean ± s.d. from three experimental replicates (*P ⩽0.01).

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Western Blot, Control

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 7. Effect of PAK4 downregulation on orthotopic tumor growth in nude mice. (a) Paraffin-embedded brain tumor sections were stained and tumor volumes were measured as described in Materials and methods section. Relative tumor size is shown as mean ± s.d. obtained from different groups as indicated (n = 6; *P ⩽0.05, **P ⩽0.01). (b) Immunohistochemical analysis of brain tumors from nude mice that were intracranially implanted with SV or PAK4sh cells and subjected to IR treatments as described in Materials and methods section; representative micrographs are shown. Inset: staining with Non-specific IgG. (c) Confocal microscopy was performed in tumor sections to determine N-cadherin (red) and E-cadherin expression (green) levels. (d) Schematic diagram represents the radiation-induced PAK4 nuclear translocation, binding with PPARγ and co-recruitment of PAK4/PPARγ complex on to Nox1 promoter, which further results in Nox1 transactivation, ROS generation and EMT induction in glioma cells.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Staining, Immunohistochemical staining, Confocal Microscopy, Expressing, Translocation Assay, Binding Assay