Journal: Journal of cell science

Article Title: alpha6beta4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-kappaB-dependent resistance to apoptosis in 3D mammary acini.

doi: 10.1242/jcs.03484

Figure Lengend Snippet: Fig. 2. Rac activity is necessary for resistance to apoptosis in 3D mammary acini. (A) FACS analysis showing increased EGFP expression in MECs expressing the EGFP-tagged N17 Rac (P4) in comparison with vector control MECs (P3). (B) Representative immunoblot of GTP-Rac, Rac and E-cadherin in vector control MECs grown as 2D monolayers in comparison with MECs expressing EGFP-tagged N17 Rac. The data illustrate that N17Rac significantly reduces GTP-Rac levels in MECs. (C) Average relative specific activity of Rac in MECs calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization of data illustrated in B. (D) Representative immunoblot of phospho-Pak1 and total Pak1 in 2D monolayer cultures of control MECs and MECs expressing EGFP- tagged N17Rac demonstrating how loss of Rac activity also reduces Pak1 activity. (E) Bar graph depicting the average degree of reduction of Pak1 activity in MECs expressing EGFP-N17Rac in comparison with control MECs. (F) Dose-response curves of the percentage apoptosis, as determined by calculating the number of activated caspase-3-positive cells divided by the total cell number, showing how 3D rBM polarized mammary acini with reduced Rac activity are now more sensitive to both chemotherapeutic (taxol) and receptor-mediated (Trail) apoptotic stimuli. Results are the mean±s.e.m. of three to five separate experiments. *P0.05 (C,E,F); **P0.01 (F).

Article Snippet: The sources of the primary antibodies used in these studies were as follows: 4 integrin, clone 3E1 (Chemicon); phospho-Bad (Ser136), Bad, cleaved caspase 3, phospho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak 3, Pak 4, phospho-Pak4 (Ser474)/Pak5 (Ser602)/Pak6 (Ser560), rabbit sera (all from Cell Signaling); E-cadherin, clone 36, and Rac1, clone 102 (BD Biosciences); HA.11, clone 16B12 (Covance); and NF- B p65, rabbit sera (Santa Cruz Biotechnology).

Techniques: Activity Assay, Expressing, Comparison, Plasmid Preparation, Control, Western Blot

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1 was significantly upregulated and acted as an oncogene in GBM. (A) The kinase activity of PAK1 in GBM cells and normal human astrocytes (HA). (B) The transcriptomic expression of PAK1 in the COSMIC project. (C) The protein level of PAK1 in glioma cells. (D) In the Human Protein Atlas, the representative IHC staining of PAK1 in low-grade and high-grade gliomas was presented. (E) The PAK1 staining in lower grade glioma (WHO II and III) is lower than that in GBM samples. (F) CCK-8 assay indicated that the proliferative potential of LN229 cells was attenuated in PAK1 shRNA groups. (G) The 7 T MR images indicated that the tumor cell growth was inhibited in mice with PAK1 knockdown. Scale bars: 4 mm. (H) The IHC staining of MKI67 in PAK1 shRNA transfected and control tumors in xenograft mice. (I) The prognostic implication and expression pattern of PAK1 in human GBM CGGA data. The OS of GBM patients was shown. The level of PAK1 was higher in the MES subtype compared to Classical or Proneural subtypes

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Activity Assay, Expressing, Immunohistochemistry, Staining, CCK-8 Assay, shRNA, Knockdown, Transfection, Control

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: Bioinformatic analysis in GBM patients reveals an important relationship between PAK1 and autophagy. The cellular component (A) and KEGG pathway (B) for PAK1-positively-associated genes. (C) The annotation indicated that PAK1 was positively associated with several biological processes mainly involved in immune-related processes, protein modification, metabolic processes, cell death, and particularly autophagy. (D) The heatmap of autophagy-related genes (blue-red), pathway, and relevant clinical features

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Modification

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1 promotes autophagy which is needed for PAK1-enhanced GBM cell proliferation. (A) U87 and U118 cells were transfected with or without PAK1 construct in combination with or without increasing concentrations of 3-MA (1 and 10 mM). Cells were subjected to hypoxia, the relative number of cells was quantified using MTT assay. Each sample was performed in triplicate. (B) U87 cells were overexpressed with or without PAK1, together with or without siRNA targeting ATG5 . Cells were subjected to hypoxia. Relative cell numbers were then quantitated as in A . Immunoblotting assay (left panel) and qRT-PCR (right panel) using lysates from transfected U87 cells was performed to demonstrate ATG5 knockdown and assess autophagy. (C) U87 and U118 cells were transfected with a control vector or PAK1 construct. Then cells were subjected to hypoxia, with or without treatment of BafA 1 . Western blots were performed as indicated (left panel) and qRT-PCR (right panel) to demonstrate PAK1 overexpression. (D and E) LN229 cells were transfected with PAK1 shRNA-#1 or -#2, respectively. Then cells were subjected to hypoxia, with or without treatment of BafA 1 . qRT-PCR was performed to demonstrate PAK1 knockdown ( D lower panel). The level of LC3B-II was shown ( D , upper panel). Representative figure of GFP-LC3 puncta ( Eii ) and the quantification of autophagosomes ( Ei , data was shown as the mean ± SD of 150 cells) was shown. Scale bar: 0.05 mm

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Transfection, Construct, MTT Assay, Western Blot, Quantitative RT-PCR, Knockdown, Control, Plasmid Preparation, Over Expression, shRNA

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1 directly phosphorylates ATG5 at conserved T101 residue in GBM cells. (A) Volcano plot indicating the interactors of PAK1 in U87 cells. (B) Hypoxia-induced the co-immunoprecipitation of endogenous PAK1 and ATG5 in LN229 cells. (C) PLA assay indicated that hypoxia promoted the cytoplasmic co-localization of endogenous PAK1 and ATG5 in LN229 cells. Scale bar: 0.01 mm. (D) Ni-NTA agarose beads were used to immobilize bacterially purified His-PAK1 proteins, then incubated with HA-ELP3 and Ac-CoA. Then these beads were incubated with purified GST-ATG5 and [γ- 32 P] ATP kinase buffer. Autoradiography was performed. (E) Mass spectrometric analysis was performed to identify the PAK1-induced phosphorylation site of ATG5. (F) Ni-NTA agarose beads were used to immobilize bacterially purified His-PAK1 proteins as indicated. Then these beads were incubated with GST-ATG5 or GST-ATG5 T101A mutant and [γ- 32 P] ATP kinase buffer. ATG5 phosphorylation was examined. (G) LN229 cells with Flag-PAK1 transfection were submitted to hypoxia treatment or not. Immunoprecipitation (IP) with anti-Flag was performed. A specific anti-p-ATG5 (T101) antibody produced by our group was used to detect ATG5 phosphorylation. (H) LN229 cells with or without sh PAK1 or WT HA-PAK1 or HA-PAK1 K420 R transfection were subjected to hypoxia. A specific anti-p-ATG5 (T101) antibody produced by our group was used to detect ATG5 phosphorylation. (I) Before hypoxia treatment, PAK1-depleted LN229 cells with the reintroduction of WT HA-PAK1 or HA-PAK1 K229A mutant were subjected to FRAX597 treatment or not for 1 hr. ATG5 (T101) phosphorylation was measured as indicated

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Residue, Immunoprecipitation, Purification, Incubation, Autoradiography, Phospho-proteomics, Mutagenesis, Transfection, Produced

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1-induced phosphorylation on T101 of ATG5 inhibits ATG5 ubiquitination and promotes its stability. (A) The levels of ATG5 mRNA in U87 cells or LN229 cells with indicated treatment were measured using qRT-PCR. (B and C) The effect of treatment of CHX (20 μg/ml) on the half-life of ATG5 protein in LN229 or U87 cells with indicated treatment, respectively. (D) The effect of MG-132 on ATG5 degradation in LN229 cells with PAK1 deficiency. (E) The effect of different ATG5 constructs on the levels of ATG5 protein in HEK293 T cells and LN229 cells, respectively. (F) The effect of ATG5 (T75) mutants on the expression of ATG5 protein. (G) The effect of CHX treatment on the levels of ATG5 protein in HEK293 T cells transfected with different ATG5 plasmids. (H) The effect of different ATG5 plasmids on the level of ATG5 ubiquitination in HEK293 T cells. We used an anti-Flag antibody to check the protein level of ATG5 in E-H.

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Quantitative RT-PCR, Construct, Expressing, Transfection

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1-mediated phosphorylation of ATG5 protein at T101 promotes ATG12–ATG5-ATG16L1 binding and autophagosome in response to hypoxia in GBM cells. ATG16L1 was immunoprecipitated from LN229 cells pretreated with a PAK1 inhibitor and then exposed to hypoxia (A) or transfected with control or PAK1 shRNA with or without hypoxia (B). Cells were treated with MG132 before harvest. Immunoblotting confirmed ATG12–ATG5 presence in immunocomplexes. (C) Stably expressed HA-PAK1 or HA-PAK1 K229A U87 cells were transfected with Flag-ATG12, Flag-ATG5 (labeled as Flag-ATG12/ATG5), and His-ATG16L1. His-ATG16L1 was immunoprecipitated, and immunocomplexes were analyzed by western blot with the indicated antibodies. (D) Stably expressed HA-PAK1 U87 cells were transfected with Flag-ATG12, Flag-ATG5 (labeled as Flag-ATG12/ATG5), or Flag-ATG5 101A (labeled as Flag-ATG12/ATG5 101A ), and His-ATG16L1. His-ATG16L1 was immunoprecipitated and western blots were performed as indicated. (E) The endogenous ATG5 in LN229 cells was knocked down or not. Then WT Flag-ATG5 or ATG5 T101A or ATG5 T101D were reintroduced into LN229 cells which were cultured with or without hypoxia treatment. Western blots were performed with anti-SQSTM1 or LC3B antibody. (F) The cells in ( E ) were transfected with GFP-LC3 and representative images were shown. Scale bar: 0.01 mm

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Phospho-proteomics, Binding Assay, Immunoprecipitation, Transfection, Control, shRNA, Western Blot, Stable Transfection, Labeling, Cell Culture

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1 at K420 acetylation promotes the binding of PAK1 to ATG5 and autophagy initiation in GBM cells upon hypoxia. (A) LN229 and U251 cells were treated with 30 μM anacardic acid (AA) and subjected to hypoxia or normoxia for one day. Then the indicated antibodies were probed to analyze the cell lysates. (B) MS identified the site of acetylation of PAK1 in hypoxia-treated GBM cells. (C) Ni-NTA agarose beads were used to immobilize bacterially purified WT His-PAK1 or His-PAK1 K420 R proteins, then incubated with HA-ELP3 and Ac-CoA. Then these beads were incubated with purified GST or GST-ATG5 to perform His bead pull-down assay. (D) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1 K420 R mutant and were subjected to hypoxia or not. Then immunoprecipitation analysis with indicated antibodies was performed. (E) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1, or Flag-PAK1 K420 R , and were subjected to hypoxia or not. Western blots were performed as indicated. (F) GFP-LC3 was transiently expressed in PAK1-depleted LN229 cells which were reintroduced with WT Flag-PAK1 or Flag-PAK1 K420 R or Flag Flag-PAK1 K420Q . Representative pictures were presented and quantitation of GFP-LC3 puncta from 10 different images was performed. Scale bar: 0.01 mm. (G) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1 K420Q mutant were subjected to hypoxia or not. Then immunoprecipitation analysis with indicated antibodies was performed. (H) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1 K420Q and were subjected to hypoxia or not. Western blots were performed as indicated

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Binding Assay, Purification, Incubation, Pull Down Assay, Mutagenesis, Immunoprecipitation, Western Blot, Quantitation Assay

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: ELP3 is identified as an acetyltransferase of PAK1 in response to hypoxia. (A) One siRNA library with two siRNAs against each of 19 human KAT genes was generated. Each siRNA was transiently transfected into LN229 cells, and the mRNA level of KAT genes was determined by quantitative real-time PCR (left panel). LN229 cells transfected as described were subjected to hypoxia and then harvested for immunoprecipitation with PAK1. Then western blotting was used to detect the acetylation level of PAK1. Band intensity of Ac-K was quantified (right panel). (B) Two different siRNAs targeting ELP3 were transiently transfected into hypoxia-treated LN229 cells. The acetylation level of endogenous PAK1 was determined. (C) ELP3 overexpression increased PAK1 acetylation. Indicated plasmids were transiently co-overexpressed in U87 cells under hypoxia, and PAK1 protein was purified by IP, the acetylation level was determined by western blot. (D) PAK1 with an increasing dose of ELP3 were co-transfected into U87 cells under hypoxia. Acetylation assessment of PAK1 was performed with an Ac-K antibody. (E) Association of endogenous ELP3 with endogenous PAK1 in LN229 cells. PAK1 was immunoprecipitated from LN229 cells treated with or without hypoxia, and the precipitates were analyzed using anti-ELP3. (F) In vitro acetylation assays using purified GST-PAK1 and HA-tagged WT-ELP3 or ELP3 Y529A . (G) Flag-tagged PAK1 or PAK1 K420 R mutant was expressed in U87 cells with ELP3 overexpression. Acetylation assessment was performed with a special PAK1-K420-Ac antibody produced by this group. (H) Ni-NTA agarose beads were used to immobilize purified WT His-PAK1 or His-PAK1 K420 R . Then these beads were mixed with or without Acetyl-CoA and WT Flag-ELP3, GST or GST-ATG5 to perform the affinity-isolation assay. (I) The endogenous ELP3 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-ELP3 or Flag-ELP3 Y529A mutant were cultured under hypoxia for 30 min. Immunoprecipitation analysis was performed

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Generated, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Over Expression, Purification, In Vitro, Mutagenesis, Produced, Isolation, Cell Culture

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: ELP3-mediated K420 acetylation activates PAK1 by decreasing PAK1 dimerization and promoting T423 phosphorylation. (A) WT ELP3, but not its inactive Y529A mutant, suppressed the association of differently tagged PAK1 subunits. WT HA-ELP3 or its catalytic-inactive mutant Y529A was co-expressed with GFP- and Flag-PAK1 in HEK293 cells. Western blot was used to determine the interaction between Flag- and GFP-PAK1. (B) ELP3 co-expression in U87 cells decreased the association of differently tagged WT-PAK1 but not the K420R mutant. Flag- and GFP-tagged PAK1 (WT or K420 R mutant) were co-expressed with or without HA-tagged ELP3. Western blot was used to determine the interaction between Flag- and GFP-PAK1. (C) ELP3 was stably knocked down in LN229 cells using two independent shRNAs. Then these cells were subjected to 0.025% glutaraldehyde treatment. The endogenous PAK1 was immunoprecipitated from LN229 cells, and western blots were used to analyze the levels of monomeric and dimeric PAK1. (D) ELP3 was stably knocked down in LN229 cells. Then these cells were reintroduced with WT-ELP3, or its Y529A mutant and subjected to 0.025% glutaraldehyde treatment. The endogenous PAK1 was immunoprecipitated from LN229 cells, and western blots were used to analyze the levels of monomeric and dimeric PAK1. (E) U87 cells were transfected with WT Flag-PAK1 or PAK1 K420 R and then subjected to hypoxia. Western blots were performed using indicated antibodies. (F) U87 cells were transfected with ELP3 shRNA and then subjected to hypoxia. Western blots were performed to analyze the T423 phosphorylation of Flag-PAK1. (G and H) In vitro phosphorylation assay. Flag-PAK1 was immunoprecipitated with an anti-Flag antibody from cell extracts in (E and F). Then myelin basic protein (MBP) was used to examine the kinase activity of PAK1 as a phosphorylation substrate. (I) the acetylation levels of K420 of WT Flag-PAK1 or PAK1 T423A were determined using immunoblots in hypoxia-treated U87 cells or LN229 cells with or without PDK1 knockdown

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Expressing, Stable Transfection, Immunoprecipitation, Transfection, shRNA, In Vitro, Activity Assay, Knockdown

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: SIRT1 deacetylated PAK1 at K420 and suppressed autophagy in GBM cells. (A) The indicated constructs were transfected into U87 cells which then were treated with hypoxia. The lysates of U87 cells were incubated and immunoprecipitated with Flag beads. Anti-acetyl-lysine (Ac-K) antibody was probed to detect the acetylation of PAK1. (B) Anti-Ac-K antibody was used to perform immunoprecipitation in LN229 cells with or without SIRT1 knockdown. Western blot with anti-PAK1 antibody was performed to detect PAK1 acetylation. (C) Flag beads were used to perform the immunoprecipitation in hypoxia-treated U87 cells with Flag-PAK1 overexpression. Purified WT GST-SIRT1 or GST-SIRT1 363A protein then was mixed with the immunoprecipitated complex at 4°C for 4 h, which was subjected to the treatment of nicotinamide (NAM, 200 μM) or nicotinamide adenine dinucleotide (NAD, 50 μM). Then samples were analyzed with immunoblotting to determine the relative levels of PAK1 acetylation. (D) Indicated constructs were overexpressed in U87 cells which then were subjected to hypoxia. U87 cells were lysed and the relative PAK1 (K420) acetylation levels were determined with a special PAK1-K420-Ac antibody. (E) U87 cells were treated with 200 μM NAM for 6 h. Hypoxia-treated U87 cells were lysed and the relative PAK1 (K420) acetylation levels were determined with a special PAK1-K420-Ac antibody. Then the relative expression ratio of PAK1-K420-Ac and PAK1 was quantified as indicated. (F) U87 cells were treated by indicated compound and plasmid, autophagy was examined. Scale bar: 0.01 mm

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Construct, Transfection, Incubation, Immunoprecipitation, Knockdown, Western Blot, Over Expression, Purification, Expressing, Plasmid Preparation

Journal: Autophagy

Article Title: Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

doi: 10.1080/15548627.2020.1731266

Figure Lengend Snippet: PAK1-mediated ATG5 phosphorylation at T101 promotes GBM development and relates to a poor prognosis. (A) The effects of PAK1 or ATG5 knockdown and reconstituted expression of the indicated plasmids on the proliferation of LN229 cells cultured in hypoxia condition for 3 d using MTT assay. (B and C) The effects of PAK1 or ATG5 knockdown with or without reintroduction of indicated plasmids on GBM growth after LN229 cells were injected into intracranially nude mice (n = 10 per group). And representative images (scale bar, 100 μm) of H&E and tumor volumes were shown. (D) IHC staining of human GBM samples. Scatterplot depicting the indicated levels of PAK1-K420-Ac and p-ATG5 (T101) protein in human GBM (n = 88) by IHC (bottom). Pearson’s correlation coefficient was determined. (E) GBM patients were stratified by ATG5 (T101) phosphorylation (top) or PAK1 (K420) acetylation (bottom). And OS was determined using Kaplan-Meier plots

Article Snippet: WT-PAK1, PAK1 T423E , ATG5 , and ATG16L1 plasmids were from Addgene: WT-PAK1 and PAK1 T423E (Addgene, 12,209 and 12,208; deposited by Jonathan Chernoff), ATG5 (Addgene, 22,948; deposited by Noboru Mizushima), FLAG-ATG16L1 constructs (Addgene, 24302; deposited by Noboru Mizushima).

Techniques: Phospho-proteomics, Knockdown, Expressing, Cell Culture, MTT Assay, Injection, Immunohistochemistry

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: bioRxiv

Article Title: Control of OPC proliferation and repopulation by the intellectual disability gene PAK1 under homeostatic and demyelinating conditions

doi: 10.1101/2024.04.26.591153

Figure Lengend Snippet: A , Western blot (WB) assay of PAK1 and p-PAK (Thr423) in mouse brain, P, postnatal day. B , WB assay of PAK1 in P15 brain C , IHC of Sox10 and PAK1 on paraffin sections of P15 brain D , IHC of Sox10 and eGFP on frozen sections of P15 brain E , WB time course assay of PDGFRa, MBP, PAK1, and p-PAK1 (Thr423) in primary rat OPC (day 0), and differentiating OL at day 1, 2, 3, and 7. F-G , quantification of PAK1 and p-PAK1 protein levels (statistical parameters, see Table S1, hereafter) H , ICC of PAK1 with OPC marker PDGFRa at day 0 and OL marker MBP at day 4 of rat OLs I, ICC of p-PAK1 (Thr423) with PDGFRa and MBP. J , Gene ontology biological process (GO_BP) terms of PAK1’s interacting proteins in primary rat OPCs (see Table S2-4). K, heatmap of PAK1’s interacting proteins overrepresented in the GO_BP of RNA processing and cell cycle. Scale bars=10µm.

Article Snippet: Specifically, cells were transfected with either 2 μg of an empty vector, 2 μg of a constitutively active PAK1 plasmid (pCMV6M-PAK1 T423E, Addgene plasmid # 12208 by Jonathan Chernoff), or 2 μg of a dominant-negative PAK1 plasmid (pCMV6M-PAK1 H83L H86L K299R, Addgene plasmid # 26592 by Jonathan Chernoff).

Techniques: Western Blot, Marker

Journal: bioRxiv

Article Title: Control of OPC proliferation and repopulation by the intellectual disability gene PAK1 under homeostatic and demyelinating conditions

doi: 10.1101/2024.04.26.591153

Figure Lengend Snippet: A, experimental designs for panels B-C. B , total numbers of OPCs after 4 days of growth C, representative confocal images and quantification of EdU + and Ki67 + proliferating OPCs D , experimental design for panel E-H E , WB assay of PAK1 and p-PAK1 (Thr423) at day 4 of OPC expansion F , total numbers of OPCs after 4 days of growth G, percentage of Ki67 + proliferating OPCs and EdU + OPCs among total PDGFRa + OPCs H, representative confocal image and quantification of cleaved caspase 3 (CC3), Sox10, and PDGFRa I , fold enrichment of PDGFRa in Co-IP by PAK1 antibody and IgG control J , Co-IP followed by immunoblot (IB) assay of PDGFRa-PAK1 interaction in OPCs K , WB assay of protein extracted from rat OPCs transfected with Ctrl, constitutive active, and dominant negative (inactive) PAK1 for 48 hours. L , quantification of p-PDGFRa at Y754 and Y849 versus total PDGFRa protein level M , representative confocal images of PDGFRa and Ki67 in rat OPCs at 48 hours after transfection N , quantification of percentage (left) and density (# per mm 2 , right) of Ki67 + PDGFRa + proliferating OPCs Scale bar: C, 100 µm; H, M, 50 µm.

Article Snippet: Specifically, cells were transfected with either 2 μg of an empty vector, 2 μg of a constitutively active PAK1 plasmid (pCMV6M-PAK1 T423E, Addgene plasmid # 12208 by Jonathan Chernoff), or 2 μg of a dominant-negative PAK1 plasmid (pCMV6M-PAK1 H83L H86L K299R, Addgene plasmid # 26592 by Jonathan Chernoff).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Transfection, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Control of OPC proliferation and repopulation by the intellectual disability gene PAK1 under homeostatic and demyelinating conditions

doi: 10.1101/2024.04.26.591153

Figure Lengend Snippet: A , experimental designs of Cre-loxP-mediated PAK1 conditional knockout (cKO) constitutively in Sox10-expressing OPCs. Pak1 cKO - Sox10-Cre:Pak1 fl/fl , Pak1 Ctrl – Sox10-Cre:Pak1 +/+ , Panel C-F B , RT-qPCR (left) and WB assays of Pak1 mRNA and protein in P7 forebrain C-E , representative confocal images and quantification of PDGFRa + OPCs and phosphor histone H3 (PH3) + PDGFRa + mitotic OPCs in the brain subcortical white matter (SCWM). G , experimental designs of tamoxifen-inducible PAK1 cKO for Panel H-K. Pak1 cKO - Pdgfra-CreER T2 :Pak1 fl/fl , Pak1 Ctrl – Pdgfra-CreER T2 :Pak1 +/+ H-I , representative confocal image and quantification of PDGFRa + OPCs, EdU + / PDGFRa + dividing OPCs, and percentage among total OPCs in the brain SCWM. J , representative confocal images, density of Ki67 + PDGFRa + proliferating OPCs, and percentage among total OPCs in the brain SCWM. K , representative confocal images, density of PH3 + PDGFRa + mitotic OPCs, and percentage among total OPCs in the brain SCWM. L , experimental designs of tamoxifen-inducible PAK inhibition (peptide inhibition domain, PID) for Panel M-T. Cre Ctrl - Pdgfra-CreER T2 , Cre PID – Pdgfra-CreER T2 :LSL-PID. Tamoxifen was i.p. administered to neonatal mice at P1, P2, P3. EdU was i.p. injected 2 hours prior to sacrifice at P7. PID expression is concomitant with EGFP expression in PDGFRa + OPCs. M , WB assay of p-PAK1 (Thr423) and total PAK1 in the P7 brain. Beta-actin, internal protein loading control. N , EGFP expression in the SCWM of Cre:PID and Cre Ctrl mice. O-P , representative confocal images and densities (#/mm 2 ) of PDGFRa + total OPCs and Ki67 + PDGFRa + proliferating OPCs in the brain SCWM. Q-R , representative confocal images and density (#/mm 2 ) and percentage of EdU + PDGFRa + proliferating OPCs in the brain SCWM. S-T , representative confocal images and density (#/mm 2 ) and percentage of PH3 + PDGFRa + proliferating OPCs in the brain SCWM. Scale bars=50 µm.

Article Snippet: Specifically, cells were transfected with either 2 μg of an empty vector, 2 μg of a constitutively active PAK1 plasmid (pCMV6M-PAK1 T423E, Addgene plasmid # 12208 by Jonathan Chernoff), or 2 μg of a dominant-negative PAK1 plasmid (pCMV6M-PAK1 H83L H86L K299R, Addgene plasmid # 26592 by Jonathan Chernoff).

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Inhibition, Injection, Control

Journal: bioRxiv

Article Title: Control of OPC proliferation and repopulation by the intellectual disability gene PAK1 under homeostatic and demyelinating conditions

doi: 10.1101/2024.04.26.591153

Figure Lengend Snippet: A , experimental designs for panel B-G. P60 transgenic mice carrying Pak1 cKO, PID, and respective Cre Ctrl were i.p. injected with 5-day tamoxifen. After 2 weeks clearance time, treated mice were induced focal demyelination by lysolecithin (lysophosphatidylcholine LPC) stereotaxic injection into the corpus callosum and killed at 5 days post-lesioning (dpl), a time point of active OPC proliferation and lesion recruitment. Neural red (NR) was i.p. injected 2 hours prior to sacrifice to track lesions. B-D . confocal images ( B ) and quantification of PDGFRa + OPCs ( C ), EdU + PDGFRa + dividing OPCs and percentage ( D ) in lesion cores of Pdgfra-CreER T2 :Pak1 fl/fl (Pak1 cKO) versus Pdgfra-CreER T2 control mice. E-H . confocal images ( E ) and quantification of PDGFRa + OPCs ( F ), EdU + PDGFRa + dividing OPCs ( G ) and percentage ( H ) in lesion cores of Pdgfra-CreER T2 :LSL-PID (Pak inhibition) versus Pdgfra-CreER T2 control mice.

Article Snippet: Specifically, cells were transfected with either 2 μg of an empty vector, 2 μg of a constitutively active PAK1 plasmid (pCMV6M-PAK1 T423E, Addgene plasmid # 12208 by Jonathan Chernoff), or 2 μg of a dominant-negative PAK1 plasmid (pCMV6M-PAK1 H83L H86L K299R, Addgene plasmid # 26592 by Jonathan Chernoff).

Techniques: Transgenic Assay, Injection, Control, Inhibition

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-PAK1/2/3 (rabbit polyclonal) , Cell Signaling Technology , #2604 , WB (1:1000).

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy