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: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: NecroMφ-EVs shuttled Pak4 to incur osteogenic changes of TSPCs in vitro and traumatic HO formation in vivo. a Proteomics sequencing was performed between EVs derived from PBS&DMSO stimulated BMDMs (ConMφ-EVs) and necroptotic BMDMs (NecroMφ-EVs) in vitro and between sham group and the tendon lesions at 7 days in vivo. Intersection Venn diagram was drawn, criterion: P value < 0.05, fold-change >2, upregulation. After further intersecting with in vivo transcriptomics sequencing between sham group and the tendon lesions at 7 days (criterion: upregulation), the fold-change of finally 113 shared expressed proteins from highest to lowest was drawn (detailed in Fig. ). b IF staining was used to detect colocalization of Pak4 (red) and Cd63 (green) in BMDMs between normal group and necroptosis induction; n = 6, scale bar = 50 μm (original magnification) and 12.5 μm (insert magnification of the boxed area, 4.0x). c IF staining was used to detect colocalization of Pak4 (red) and Cd63 (green), co-localized with F4/80 (purple), between sham group and the tendon lesions at 7 days; n = 5, scale bar = 50 μm (original magnification) and 6.25 μm (insert magnification of the boxed area, 8.0x). d ALP and ARS staining were used to detect the osteogenesis of TSPCs in addition of NecroMφ-EVs or PAK4 −/− - EVs; n = 6. Quantification was shown in Fig. . e Micro-CT was used to detect HO formation between the tendon lesions at 10 weeks from Flox mice and Pak4 cKO mice, as well as in addition of NecroMφ-EVs or PAK4 −/− - EVs; n = 5. Quantification was shown in Fig.

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: In Vitro, In Vivo, Sequencing, Derivative Assay, Staining, Micro-CT

Journal: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: Fatty acid β-oxidation (FAO) was responsible for the biological effects of Pak4 from NecroMφ-EVs on the osteogenic behavior of TSPCs in vitro and traumatic HO formation in vivo. a High-throughput whole-transcriptome sequencing was performed and showed by Wikipathways enrichment (top 10) and heatmap (FAO), between the sham group and tendon lesions at 7 days; n = 3. b High-throughput whole-transcriptome sequencing was performed and showed by GSEA for “fatty acid beta-oxidation” and “fatty acid beta-oxidation using acyl-CoA dehydrogenases” between the osteogenic induced TSPCs, in addition of NecroMφ-EVs or PAK4 −/− - EVs in vitro. c Metabolites analyses for palmitoyl-CoA and acetyl-CoA were performed to detect the FAO in osteogenic induced TSPCs, in addition of NecroMφ-EVs or PAK4 −/− - EVs; n = 6, **** P < 0.000 1. d IF staining was used to detect the positive cells of Lcad (red), co-localized with Pdgfr-α (green), between the tendon lesions at 7 days from Flox mice and Pak4 cKO mice, as well as in addition of NecroMφ-EVs or PAK4 −/− - EVs; n = 5, ** P < 0.01, scale bar = 100 μm (original magnification) and 12.5 μm (insert magnification of the boxed area, 4.0x). e ALP and ARS staining were used to detect the osteogenesis of TSPCs in addition of PAK4 −/− - EVs, with or without sh-Lcad ; n = 6, **** P < 0.000 1. f Micro-CT was used to detect HO formation in the tendon lesions at 10 weeks in addition of PAK4 −/− - EVs, with or without sh-Lcad ; the volume of HO was quantified from each group; n = 5, **** P < 0.000 1

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: In Vitro, In Vivo, High Throughput Screening Assay, Sequencing, Staining, Micro-CT

Journal: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: Pak4 from NecroMφ-EVs directly binds to Fabp3 at S122 phosphorylation site in TSPCs in burn/tenotomy mice. a Mass spectrometry analysis was used to detect the potential molecules that could bind with Pak4 between the sham group and tendon lesions at 7 days; n = 3. Intersection Venn diagram was drawn, criterion: identified in all three biological samples. The top 20 Score Sequest HT of finally 134 molecules (capable of directly binding to Pak4 in the BTT group but not in the control group) in 3 individual BTT group from highest to lowest was drawn. b IF staining was used to detect the colocalization by the positive cells of Pak4 (red) and Fabp3 (green), co-stained with DAPI (blue), in the osteogenic induced TSPCs, in addition of PBS or NecroMφ-EVs; n = 6, scale bar = 50 μm (original magnification) and 12.5 μm (insert magnification of the boxed area, 4.0x). c Co-IP analysis was used to verified the bind relationship between Pak4 and Fabp3 in the osteogenic induced TSPCs, in addition of PBS or NecroMφ-EVs; n = 3. d Phosphorylation prediction was used to predict the murine phosphorylation site of Pak4 for Fabp3. Mass spectrometry analysis was used to detect the potential phosphorylation site of Pak4 for Fabp3 in the osteogenic induced TSPCs in addition of NecroMφ-EVs or PAK4 −/− - EVs. e IF staining was used to detect the expression of p-Fabp3 (red), co-stained with phalloidin (green) and DAPI (blue) in the osteogenic induced TSPCs, in addition of NecroMφ-EVs or PAK4 −/− - EVs; n = 6, scale bar = 100 μm (original magnification) and 25 μm (insert magnification of the boxed area, 4.0x). f WB analysis was used to detect the levels of p-Fabp3 in the osteogenic induced TSPCs in addition of NecroMφ-EVs or PAK4 −/− -EVs; n = 3, **** P < 0.000 1

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: Phospho-proteomics, Mass Spectrometry, Binding Assay, Control, Staining, Co-Immunoprecipitation Assay, Expressing

Journal: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: Phosphomimetic mutant of Fabp3 on S122 site reduced FAO of TSPCs and increased traumatic HO formation in burn/tenotomy mice. a Metabolites analyses for palmitoyl-CoA and acetyl-CoA were performed to detect the FAO in osteogenic induced TSPCs, in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 6, **** P < 0.000 1. b Seahorse test was used to detect the oxidative phosphorylation level in the osteogenic induced TSPCs in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 3, represented PAK4 -/- & S122 group, and represented PAK4 −/− group, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significant difference. c ALP and ARS staining were used to detect the osteogenesis of TSPCs in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 6. d IF staining was used to detect osteogenesis by the positive cells of Runx2 (orange) in the tendon lesions at 3 weeks in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 5, scale bar = 100 μm (original magnification), **** P < 0.000 1. e SOFG staining was used to detect osteogenesis region in the tendon lesions at 3 weeks in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 5, scale bar = 200 μm (original magnification). f Micro-CT was used to detect HO formation in the tendon lesions at 10 weeks in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; the volume of HO was quantified from each group; n = 5, **** P < 0.000 1. g HE staining was used detect ossification region in the tendon lesions at 10 weeks, in addition of PAK4 −/− - EVs, with or without Fabp3 S122D ; n = 5, scale bar = 200 μm (original magnification)

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: Mutagenesis, Phospho-proteomics, Staining, Micro-CT

Journal: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: PAK4 and FABP3-related FAO were found to be in an activated state in the human HO tissues. a Schematic depiction of human traumatic HO tissues. b H&E staining was used to detect ossification region between HO tissue section and normal tendon tissue section; n = 5, scale bar = 50 μm (original magnification). c IF staining was used to detect the positive cells of PAK4 (red), p-FABP3 (red), and LCAD (red) between HO tissue section and normal tendon tissue section; n = 5, scale bar = 50 μm (original magnification)

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: Staining

Journal: Bone Research

Article Title: Pak4-mediated crosstalk between necroptotic macrophages and tendon stem/progenitor cells contributes to traumatic heterotopic ossification formation

doi: 10.1038/s41413-025-00463-8

Figure Lengend Snippet: Graphical summary. Following soft tissue injury, macrophages infiltrate, and necroptosis occurs. Concurrently, this process is accompanied by paracrine EVs-derived PAK4, which subsequently regulates FABP3 in TSPCs and reduces FAO. This cascade ultimately leads to osteogenic behavior in TSPCs and contributes to HO formation. Notably, PAK4 binds directly to FABP3 and phosphorylates it at the S122 site, thereby influencing FAO

Article Snippet: The p-Ripk1 (Cat# 28252-1-AP), Runx2 (Cat# 20700-1-AP), Opn (Cat# 22952-1-AP), Cd9 (Cat# 60232-1-lg), Cd81 (Cat# 27855-1-AP), Alix (Cat# 12422-1-AP), Tsg101 (Cat# 28283-1-AP), Calnexin (Cat# 10427-2-AP), Pak4 (Cat# 14685-1-AP), Eea1 (Cat# 28347-1-AP), Lcad (Cat# 17526-1-AP) and Fabp3 (fatty acid binding protein 3, Cat# 10676-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA).

Techniques: Derivative Assay

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 2. Radiation-induced nuclear localization of PAK4 and correlation with increased EMT in glioma cells. (a) PAK mRNA levels in control and IR (8 Gy)-treated cells as determined by quantitative PCR. Fold change values of PAK4 are represented as mean ± s.d. of levels obtained from at least five repetitions in three experimental replicates (*P ⩽0.01). (b) Western blotting with cellular lysates showing PAK4 and phospho- PAK4 expression in control and IR (8 Gy)-treated cells. (c) Western blot analysis of PAK4 and phospho-PAK4 levels in cytoplasmic and nuclear fractions with or without IR treatment. GAPDH and HDAC-1 were used as loading controls for cytoplasmic and nuclear fractions respectively. Relative expression levels of cytoplasmic and nuclear PAK4 were estimated by densitometry (ImageJ 1.42) and mean ± s.d. values were presented (*P ⩽0.01). (d) Immunocytochemical analysis to assess sub-cellular localization of PAK4 in control- and IR (8 Gy)-treated cells. Nuclei were counterstained with DAPI. (e) Micrographs showing morphological changes in 4910 and U251 cells after IR treatments. Scale bars: 10 μm. (f) Western blot analysis with whole-cell lysates to assess the expression of epithelial and mesenchymal regulator proteins. (g) Confocal microscopy to examine changes in N-cadherin (red) and E-cadherin (green) levels in IR-treated cells after 48 h. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (h) Assessment of total cellular ROS content in control and IR-treated cells with H2DCFDA staining as described in Materials and methods section 48 h after IR treatment. Relative ROS levels from three independent experiments are shown as mean ± s.d. (*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: Control, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Confocal Microscopy, 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 3. Association of PAK4 with PPARγ in the nuclear compartment. (a) Identification of potential PAK4 associating TFs using TF–TF Interaction Array. PAK4 was immunoprecipitated from nuclear extracts of 4910 cells with anti-PAK4 antibody and used as a bait. Isotype- specific IgG precipitates were used as negative control. PAK4 association with various TFs is identified as horizontal duplicate spots on the x-ray film. ‘+’ indicates the positive control signals. (b) 4910 cells were treated with EV and PAK4-FL for 48 h and IP experiments was performed with nuclear lysates (500 μg) from 4910 cells with specific antibodies against PAK4 and non-specific IgG followed by immunoblotting with PPARγ. Inputs indicate 10% of each pre-IP samples. (c) PPARγ IP using anti-FLAG and anti-IgG antibody from nuclear lysates of 4910 cells at 48 h post transfection with EV or FLAG-PPARγ constructs followed by immunoblotting for PAK4. (d) IP experiments using 4910 lysates with either a PAK4 specific antibody or non-specific IgG followed by immunoprobing for PPARγ. Reciprocal IPs were performed with anti-PPARγ antibody and subsequent immunoblotting with PAK4 to confirm PAK4/PPARγ association in the nucleus. (e) Identification of minimal PPARγ- interaction domain of PAK4 using bacterially expressed GST, and GST-PPARγ purified using MagneGST Pull-Down System following manufacturer’s protocol. Biotin-labeled PAK4 truncated mutants (1–290aa, 291–591aa and 1–591aa) were incubated with GST-PPARγ, separated on 10% SDS–PAGE and detected as described in Materials and methods section (Top panel). Inputs (10% samples) were analyzed by SDS–PAGE (Bottom panel). (f) Schematic representation of mapping PPARγ interacting domain on PAK4 using different truncation mutants. CRIB: Cdc42-and Rac-interactive binding motif; GID: GEF-H1 Interaction Domain; KD: Kinase Domain. (g) EMSA was performed with 4910 Nuclear extracts (5 μg) to detect PPARγ activity. For the supershift analyses, specific antibodies against PAK4 and PPARγ were incubated with control sample before adding the biotin-labeled probe.

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: Immunoprecipitation, Negative Control, Positive Control, Western Blot, Transfection, Construct, Labeling, Incubation, SDS Page, Binding Assay, Activity Assay, 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 4. Radiation-enhanced PAK4/PPARγ binding in nucleus. (a) IP with antibodies against non-specific IgG and PPARγ using nuclear lysates prepared from control and IR (8 Gy)-treated 4910 and U251 cells followed by immunoprobing with PAK4 antibody (Top panels). Reciprocal IPs were performed with anti-IgG and anti-PAK4, and subsequently immunoprobed with PPARγ to confirm changes in radiation-induced PAK4/ PPARγ interaction in these cells (Bottom panels). Representative blots from three independent experiments are shown. (b) Sub-cellular localization analyses of PAK4 (green) and PPARγ (red) by confocal microscopy in control and IR-treated cells. Scale bars: 10 μm. (c) Protein-DNA array (version II) interaction analysis performed with immunoprecipitated PAK4 from 4910 nuclear extracts as described in Materials and methods section. PAK4 binding with TF consensus sequences was detected by duplicate spots on the membrane. ‘+’ indicates positive control signal.

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: Binding Assay, Control, Confocal Microscopy, DNA Array, Immunoprecipitation, Membrane, Positive 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 5. Radiation-induced PAK4/PPARγ recruitment on the PPARγ-binding site on Nox1 promoter. (a) Real-time PCR showing Nox1 transcriptional levels in control- and IR-treated cells. The fold change values are represented as mean ± s.d. (n = 5) obtained from at least three independent experiments (*P ⩽0.01). (b) Immunoblotting shows Nox1 expression with GAPDH served as an internal control. (c) Whole-cell lysates were subjected immunoblotting and representative blots from three independent experiments were shown. (d) Schematic representation of putative PPARγ binding sites on Nox1 promoter. Seven putative PPRE sites were identified located in the promoter (4 sites), exon-1 (1 site) and intron-1 (2 sites) of human Nox1 based on analysis of a 2.9-kb 5ʹ-flanking region of Nox1 (GenBank: ABC40742.1). ChIP primers specific for R-1, R-2, R-3 and R-4 regions (blue arrows) were used to determine PPARγ recruitment on Nox1 promoter. (e) ChIP analysis of PPARγ occupancy around PPREs on the Nox1 promoter using DNA from 4910 and U251 cells and IP with anti-IgG and anti-PPARγ antibodies with and without IR treatment. 5% of pre-ChIP DNA samples served as input controls for each sample. ChIP DNA from control and IR-treated cells were analyzed by quantitative PCR using ChIP-specific primers covering Nox1 promoter regions (R-1 to R-4). ChIP amplification is shown as percent input from three different experiments (n = 5; *P ⩽0.05, **P ⩽0.01). (f) ChIP assay was performed with R-2 primers and antibodies against non-specific IgG and PPARγ using ChIP DNA as described above in both 4910 and U251 cells. Subsequently, re-ChIP assay was performed using primary ChIP amplicons with anti-IgG and anti-PAK4 antibodies; results from three experimental replicates are shown.

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: Binding Assay, Real-time Polymerase Chain Reaction, Control, Western Blot, 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

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Primer sequences used for a quantitative real-time polymerase chain reaction.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Sequencing

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Immunohistochemical expression of PAK4 and PD-L1 in human osteosarcoma tissue and survival analysis. ( a ) Immunohistochemical expression of PAK4 and PD-L1 in human osteosarcoma tissue. Original magnification, ×400. ( b ) Statistical analysis to determine cut-off points. In receiver operating characteristic curve analysis, the cut-off point for both the immunohistochemical staining scores of PAK4 expression (red arrowhead) and PD-L1 expression (blue arrow) was twelve. ( c ) Kaplan–Meier survival analysis according to PAK4 and PD-L1 expression for overall survival and relapse-free survival in 32 osteosarcoma patients. ( d ) Kaplan–Meier survival analysis for overall survival and relapse-free survival according to PAK4 and PD-L1 expression in 23 osteosarcoma patients who received postoperative chemotherapy.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Association between clinicopathologic variables and the expression of PAK4 and PD-L1 in 32 osteosarcomas.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Univariate survival analysis in 32 osteosarcomas.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques:

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Multivariate survival analysis in 32 osteosarcomas.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques:

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: The effects of PAK4 expression on proliferation and invasiveness in osteosarcoma cells. ( a , b ) MTT proliferation assays ( a ) and colony-forming assay ( b ) after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. Colony-forming assays were performed after knockdown or overexpression of PAK4 in osteosarcoma cells. U2OS (3 × 10 3 ) and KHOS/NP (3 × 10 3 ) cells were seeded in culture plates for seven days. Western blot for PAK4 and GAPDH was performed to show knockdown and overexpression of PAK4 in osteosarcoma cells. ( c , d ) Migration ( c ) and invasion ( d ) assay after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. The migration assay was performed by seeding 5 × 10 4 U2OS and 5 × 10 4 KHOS/NP cells in the upper chamber for 48 h. The invasion assay was performed by seeding 1 × 10 5 U2OS and 1 × 10 5 KHOS/NP cells in the upper chamber for 48 h. ( e ) Western blotting for PAK4, FOXO3, phosphorylated FOXO3 (pFOXO3), cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells 24 h after transfection. ( f ) Quantitative reverse-transcription polymerase chain reaction for PAK4, FOXO3, cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in osteosarcoma cells. ( g ) Gross and histologic findings of resected tumors grown in BALB/c nude mice by implanting 1 × 10 6 KHOS/NP cells that were transfected with empty vectors, shRNA for PAK4 , or plasmid for wild-type PAK4 into the marrow space of the right proximal tibia. The tumor volume was measured every seven days with the length × width × height × 0.52 mm 3 equation. The mice were euthanized six weeks after tumor implantation. Resected tumors were H&E stained. ( h ) Gross and histologic findings of pulmonary metastatic nodules in BALB/c nude mice. Arrows indicate metastatic nodules. * p < 0.05; ** p < 0.001; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4 ; shControl, control vector for shRNA; shPAK4, vector for shRNA for PAK4 .

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Migration, Invasion Assay, Transfection, Reverse Transcription, Polymerase Chain Reaction, shRNA, Plasmid Preparation, Tumor Implantation, Staining, Control

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: The effects of PAK4 expression on proliferation and apoptosis of osteosarcoma cells under treatment of doxorubicin. ( a , b ) MTT proliferation assays ( a ) and colony-forming assay ( b ) after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells treated with 0.1 µM doxorubicin. In colony-forming assay, U2OS (3 × 10 3 ) and KHOS/NP (3 × 10 3 ) cells were seeded in culture plates for one week. ( c , d ) Western blotting ( c ) and Annexin V flowcytometric analysis for apoptosis ( d ) after knockdown or overexpression of PAK4 in osteosarcoma cells under treatment with 0.1 µM doxorubicin. Doxorubicin was applied to osteosarcoma cells 24 h after transfection. * p < 0.05; ** p < 0.001; ns , not significant; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4 ; shPAK4, vector for shRNA for PAK4 ; DOX, doxorubicin.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Transfection, Plasmid Preparation, shRNA

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: PAK4 is involved in the stabilization of PD-L1 protein in osteosarcoma cells. ( a ) Western blot for PAK4 and PD-L1 after knockdown or overexpression of PAK4 in osteosarcoma cells. ( b ) Western blot for PAK4 and PD-L1 in osteosarcoma cells after treatment with PAK4 inhibitor KPT9274 (1 µM for 48 h). ( c ) Quantitative reverse-transcription polymerase chain reaction for PAK4 and PD-L1 after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. ( d ) Immunofluorescence staining of U2OS and KHOS/NP osteosarcoma cells after knockdown or overexpression of PAK4 . The cells were incubated with primary antibodies for PAK4 and PD-L1. Thereafter, the slides were incubated with Alexa Fluor 488 anti-mouse IgG (green) or Alexa Fluor 594 anti-rabbit IgG (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Images were taken with a Zeiss LSM 880 with Airyscan confocal microscope. ( e ) U2OS and KHOS/NP osteosarcoma cell lysates were immunoprecipitated with PAK4 or PD-L1 and immunoblotted with PAK4 and PD-L1. ( f ) U2OS cells were transfected with control vector or shRNA for PAK4 and treated with 30 µmol/L cycloheximide or 30 µmol/L MG132 for 0.5 to 2.0 h. Thereafter, protein lysates were immunoblotted with PD-L1 and GAPDH. ( g ) Total protein lysate from U2OS cells transfected with empty vector or shRNA for PAK4 and treated with 30 µmol/L MG132 for two hours was immunoprecipitated with PD-L1 and immunoblotted with anti-ubiquitin antibodies. ** p < 0.001; ns , not significant; CHX, cycloheximide; EV, empty vector; EVs, empty vectors; IP, immunoprecipitation; PAK4-OE, vector for wild-type PAK4 ; shControl, control vector for shRNA; shPAK4, vector for shRNA for PAK4 ; WB, western blot.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Western Blot, Knockdown, Over Expression, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Staining, Incubation, Microscopy, Immunoprecipitation, Transfection, Control, Plasmid Preparation, shRNA, Ubiquitin Proteomics

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: In vivo growth of KHOS/NP osteosarcoma cells in BALB/c nude mice and infiltrations of immune cells in C57BL/6J mice. ( a ) HOS/NP osteosarcoma cells (2.5 × 10 6 ) transfected with empty vectors, vector for shRNA for PAK4 , or vector for wild-type PAK4 were implanted subcutaneously in the back of BALB/c nude mice and grown for six weeks. Donor tumor blocks sized 2 mm × 2 mm × 2 mm from the resected tumor of BALB/c nude mice were subcutaneously implanted in the backs of C57BL/6J mice. The figure was created with BioRender.com ( https://www.biorender.com (accessed on 10 January 2024)). ( b ) Tumor growth in BALB/c nude mice. ( c ) The growth of tumor block in the back of C57BL/6J mice. Tumor volumes were calculated as “length × width × height × 0.52”. ( d ) Histologic findings and immunofluorescence staining for PAK4 (green) and PD-L1 (red) in the resected tumor grown in C57BL/6J mice. ( e ) Immunofluorescence staining and quantification for FOXP3, PD1, CD4, CD8, and CD4/CD8 ratio. Tumor cubes (0.5 mm × 0.5 mm × 0.5 mm) derived from the KHOS/NP tumor cells grown subcutaneously in BALB/c nude mice were implanted subcutaneously in C57BL/6J mice. Four days after the implantation of tumor cubes, the tumors were resected and evaluated with immunofluorescence staining for FOXP3 (green), PD1 (red), CD4 (green), and CD8 (red). The number of positively stained cells was counted in three high-power fields in each case, and the sum of the numbers was used for evaluation. The area of one high-power field image was 0.0144 mm 2 . Therefore, 0.0432 mm 2 was evaluated in each case. * p < 0.05; ** p < 0.001; ns , not significant; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4 ; shPAK4, vector for shRNA for PAK4 .

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: In Vivo, Transfection, Plasmid Preparation, shRNA, Blocking Assay, Immunofluorescence, Staining, Derivative Assay

Journal: Molecular Cancer Therapeutics

Article Title: Novel p21-Activated Kinase 4 (PAK4) Allosteric Modulators Overcome Drug Resistance and Stemness in Pancreatic Ductal Adenocarcinoma

doi: 10.1158/1535-7163.mct-16-0205

Figure Lengend Snippet: Figure 4. Impact of PAMs on PDAC CSCs. A, MiaPaCa-2 cells flow sorted for CD44þCD133þEpCAMþ according to our previously published procedure (12). In addition, MiaPaCa-2 cellswere grown for extended period of time in gemcitabine (100 nmol/L) to develop resistance cell (MiaPaCa-2 GR). RNA isolated from CSCs or MiaPaCa- 2 GR were evaluated using RT-PCR for basal expression of PAK4. B, The sorted cells were exposed to either control siRNA or PAK siRNA according to established procedures (15). The spheroid formation in PAK4 siRNA exposed CSCs was evaluated over 2 weeks, and the cell spheroids were counted and photographed under an inverted microscope (, P < 0.01 between control and PAK4 siRNA treatment groups). C, In a separate experiment, the flow-sorted CSCs were grown in ultra-low adherent 6-well plates and in spheroid-forming media DMEM/F-12 with N-2 and B-27 supplement (Invitrogen) and exposed to increasing concentrations of PAMs (0–1,000 nmol/L) twice a week for 2 weeks. The spheroids were counted under a microscope and photographed. D, MiaPaCa-2 CSCs grown in regular media were exposed to different PAMs (5 mmol/L) for 72 hours. At the end of the treatment period, RNA was isolated and RT-PCR was performed as described in Materials and Methods. Note: downregulation in stemness markers CD24, CD44, and EpCAM.

Article Snippet: PAK4 siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Inverted Microscopy, Microscopy

Journal: Cell Insight

Article Title: PAK4 phosphorylates and stabilizes MYC to promote acute myeloid leukemia

doi: 10.1016/j.cellin.2025.100274

Figure Lengend Snippet: PAK4 mediated S67 Phosphorylation Promotes AML cells proliferation. (A) Immunoblots of MYC and p-S67 MYC in primary T-ALL cells, T-ALL cell lines and AML cell lines. (B) Immunoblots of MYC in MYC depleted HL-60 cells with or without enforced expression of HA-MYC, HA-MYC S67D or HA-MYC S67A. (C) Live HL-60 cells treated in (B) were counted at the indicated time points and cell growth was plotted as shown. Data shown are means (±SD) of technical triplicates. (D) Immunoblots of MYC and p-S67 MYC in various cells treated with 10 nM AZD1152 for 24 h or 0.5 μM BMS-345541 for 12h as indicated. (E) Potential kinases phosphorylating MYC at serine 67 predicted from GPS 5.0, PhosphoSitePlus and NetPhos 3.1. (F) Immunoblots of MYC and p-S67 MYC in HL-60 cells treated with TC-DAPK6 (DAKi), Rock inhibitor-2 (ROCKi), HA-1004 (PKA/G/Ci) and KPT-9274 (PAK4i) for 2 h or 4 h. (G) Immunoblots of MYC and p-S67 MYC in HL-60 cells treated with LCH-7749944 and PF-3758309 for 1 h or 2 h. (H–I) In vitro kinase assay of recombinant GST-MYC. Active human PAK4 proteins were incubated with GST-MYC for kinase reaction. Phosphorylation was detected by immunoblots using phosphorylated protein antibodies as shown. Loading controls were shown as Coomassie blue staining in the bottom panels (H). The product of kinase reaction from (H) were treated with calf-intestinal alkaline phosphatase (CIP) for 30 min before immunoblot (I).

Article Snippet: Antibodies used are as follows: MYC (CST, #18583), PAK4 (CST, #52694), FBXW7 (Abcam, #109617), β-ACTIN (ABclonal, #AC026), GAPDH (ABclonal, #A19056), Flag-tag (Sigma-Aldrich, #F1804), HA-tag (peroxidase conjugate, Roche, #12013819001), Myc-Tag (peroxidase conjugate, ABclonal, #AE026), Ub (ABclonal, #A3207).

Techniques: Phospho-proteomics, Western Blot, Expressing, In Vitro, Kinase Assay, Recombinant, Incubation, Staining

Journal: Cell Insight

Article Title: PAK4 phosphorylates and stabilizes MYC to promote acute myeloid leukemia

doi: 10.1016/j.cellin.2025.100274

Figure Lengend Snippet: PAK4 Directly Interacts with MYC at MBⅡ domain . (A) HL-60 cell lysates were subjected to reciprocal co-IP and immunoblot to detect endogenous PAK4 and MYC interaction. IP: immunoprecipitation. (B) Lysates of HEK293FT cells overexpressing (HA-MYC and Flag-PAK4) or (HA-PAK4 and Flag-MYC) were subjected to co-immunoprecipitation (co-IP) to detect protein interaction. (C) GST pull-down to detect direct PAK4 and MYC interaction. Recombinant His-tagged PAK4 was incubated with GST-MYC in vitro before pull-down assay with GST beads, and interaction was analyzed by immunoblot. GST: glutathione-S-transferase. (D) Schematic presentation of various human MYC truncations used in PAK4-binding assays. (E) Lysates from HEK293FT cells overexpressing Flag-PAK4 and HA-MYC truncations (amino acids 1–221 and 220–439) were subjected to co-IP and immunoblot. (F) Characterization of the MB motif required for MYC and PAK4 interaction. Lysates from HEK293FT cells overexpressing HA-PAK4 and Flag-SBP-MYC truncations (amino acids 1–221 or amino acids 1–221 with Myc box domain deletion mutants (ΔMB) as shown) were subjected to co-IP and immunoblot.

Article Snippet: Antibodies used are as follows: MYC (CST, #18583), PAK4 (CST, #52694), FBXW7 (Abcam, #109617), β-ACTIN (ABclonal, #AC026), GAPDH (ABclonal, #A19056), Flag-tag (Sigma-Aldrich, #F1804), HA-tag (peroxidase conjugate, Roche, #12013819001), Myc-Tag (peroxidase conjugate, ABclonal, #AE026), Ub (ABclonal, #A3207).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Recombinant, Incubation, In Vitro, Pull Down Assay, Binding Assay

Journal: Cell Insight

Article Title: PAK4 phosphorylates and stabilizes MYC to promote acute myeloid leukemia

doi: 10.1016/j.cellin.2025.100274

Figure Lengend Snippet: PAK4 Enhances MYC Protein Stability through Phosphorylation of S67 . (A–B) PAK4 and MYC mRNA and protein were analyzed in PAK4 -depleted HL-60, OCI-AML3 and Molm13 cells by qPCR (A) and immunoblot (B). Data shown represent the means (±SD) of technical triplicates, and these experiments were independently repeated at least twice with the similar results. (C–D) Time-course analysis of MYC protein levels in PAK4 -depleted HL-60 cells (C). MYC proteins were quantified and plotted on the right (D). (E–F) Time-course analysis of HA-MYC levels in HEK293FT cells co-expressing Flag-PAK4 wild-type (WT) or kinase inactivation (KI) mutant Flag-PAK4 KI (K350A, K351A) as indicated (E). MYC proteins were quantified and plotted on the right (F).

Article Snippet: Antibodies used are as follows: MYC (CST, #18583), PAK4 (CST, #52694), FBXW7 (Abcam, #109617), β-ACTIN (ABclonal, #AC026), GAPDH (ABclonal, #A19056), Flag-tag (Sigma-Aldrich, #F1804), HA-tag (peroxidase conjugate, Roche, #12013819001), Myc-Tag (peroxidase conjugate, ABclonal, #AE026), Ub (ABclonal, #A3207).

Techniques: Phospho-proteomics, Western Blot, Expressing, Mutagenesis

Journal: Cell Insight

Article Title: PAK4 phosphorylates and stabilizes MYC to promote acute myeloid leukemia

doi: 10.1016/j.cellin.2025.100274

Figure Lengend Snippet: PAK4 depletion Promotes FBXW7 mediated MYC ubiquitylation-proteasomal degradation. (A) PAK4 -depleted HL-60 and OCI-AML3 cells were treated with MG132 (10 μM) for 6 h before harvest. PAK4, MYC and p-S67 MYC were analyzed by immunoblot, with ACTIN as a loading control. (B) Analysis of endogenous MYC polyubiquitylation. Denaturing protein IP with anti-MYC antibody and immunoblots were performed in HL-60 and OCI-AML3 cells with or without PAK4 depletion. (C) Analysis of endogenous MYC polyubiquitylation in PAK4- depleted HL-60 cells with or without enforced expression of HA-PAK4 or HA-PAK4 KI. Denaturing protein IP with anti-MYC antibody and immunoblots were performed. (D) FBXW7 was depleted by shRNA in HL-60 and OCI-AML3 cells with or without PAK4 depletion. MYC protein was analyzed by immunoblot. (E) Immunoblot analysis in HEK293FT cells co-expressing HA-MYC, Myc-FBXW7α and Flag-PAK4 as indicated. (F) Analysis of MYC polyubiquitylation. Denaturing proteins from HEK293FT cells expressing indicated constructs were pulled down with streptavidin beads and subjected to immunoblot analysis. MG132 (10 μM) was added in HEK293FT cells for 6 h before harvest. SBP: streptavidin-binding peptide. (G) Analysis of MYC polyubiquitylation. HA-tagged ubiquitin was co-transfected with Myc-FBXW7α and Flag-MYC (WT, S67A or S67D) into 293T cells. The resulting cell lysates were subjected to co-IP by anti-Flag, followed by immunoblotting of ubiquitin.

Article Snippet: Antibodies used are as follows: MYC (CST, #18583), PAK4 (CST, #52694), FBXW7 (Abcam, #109617), β-ACTIN (ABclonal, #AC026), GAPDH (ABclonal, #A19056), Flag-tag (Sigma-Aldrich, #F1804), HA-tag (peroxidase conjugate, Roche, #12013819001), Myc-Tag (peroxidase conjugate, ABclonal, #AE026), Ub (ABclonal, #A3207).

Techniques: Western Blot, Control, Expressing, shRNA, Construct, Binding Assay, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Cell Insight

Article Title: PAK4 phosphorylates and stabilizes MYC to promote acute myeloid leukemia

doi: 10.1016/j.cellin.2025.100274

Figure Lengend Snippet: Synergistic Lethality of AML Cells by Dual Targeting of PAK4 and MCL-1 . (A) PAK4 mRNA and protein were detected by qPCR (left) and immunoblots (right) in HL-60 cells. Data shown represent the means (±SD) of technical triplicates, and these experiments were independently repeated at least three times with the similar results. (B) Live HL-60 cells with or without PAK4 depletion were counted at the indicated time points and cell growth was plotted as shown. Data shown are means (±SD) of technical triplicates. (C) Live HL-60 cells treated with PAK4 inhibitor KPT-9274 were counted at the indicated time points and cell growth was plotted as shown. Data shown are means (±SD) of technical triplicates. (D–F) HA-MYC was overexpressed in HL-60 cells with PAK4 depletion. PAK4 and MYC mRNA and protein were detected by qPCR (D) and immunoblots (E). Live cells treated as described were counted at the indicated time points and cell growth was plotted as shown (F). Data shown are means (±SD) of technical triplicates. (G) Assessment of cell death in PAK4 -depleted HL-60 cells. Data shown are means (±SD) of technical triplicates. (H) Immunoblots of MYC, MCL-1, BCL2 and Bcl-XL in PAK4 -depleted HL-60 cells. (I) Immunoblots of MYC and MCL-1 in HL-60 cells treated with KPT-9274 for 72 h. (J) Assessment of cell death in HL-60 cells treated with 100 nM KPT-9274 and/or 50 nM S63845 for 48 h. Combo, combination treatment. Data shown are means (±SD) of technical triplicates.

Article Snippet: Antibodies used are as follows: MYC (CST, #18583), PAK4 (CST, #52694), FBXW7 (Abcam, #109617), β-ACTIN (ABclonal, #AC026), GAPDH (ABclonal, #A19056), Flag-tag (Sigma-Aldrich, #F1804), HA-tag (peroxidase conjugate, Roche, #12013819001), Myc-Tag (peroxidase conjugate, ABclonal, #AE026), Ub (ABclonal, #A3207).

Techniques: Western Blot