Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet: Focal cerebral ischemia increases PAK1 phosphorylation (A) Representative image of blood flow measured by laser speckle flowmeter. (B) The time-dependent changes of PAK1 and p-PAK1 in ischemic cortex at 1 h, 6 h, 12 h, or 24 h reperfusion after 1-h transient middle cerebral artery occlusion (tMCAO). The homogenates of cortical brains from tMCAO and sham treated mice were subjected to Western blot analysis using indicated antibodies. GAPDH was used as a loading control. (C and D) The quantitative analysis of immunoblotted p-PAK1 (Ser144) and PAK1 proteins. Data are expressed as mean ± SEM (n = 4). ∗p < 0.05 versus sham, one-way ANOVA with Dunnet’s post hoc test. (E) Immunofluorescence images showing the colocalization of PAK1-positive (green) with microvessel markers Lectin (red) in the ipsilateral (I)/contralateral (C) cortex at 1 h reperfusion after tMCAO. White arrow shows that PAK1 is localized on blood vessels. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Phospho-proteomics, Western Blot, Control, Immunofluorescence, Staining

Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet: PAK1 inhibition by its inhibitors or siRNA reduces the hyperpermeability of bEnd.3 endothelial monolayer in OGD model (A) Immunoblots of p-PAK1 and PAK1 with corresponding antibodies in bEnd.3 cells at reperfusion for indicated times after 2 h OGD. GAPDH was used as a loading control. (B and C) The statistical analysis of immunoblotted p-PAK1 (Ser144) and PAK1 proteins. Data are expressed as mean ± SEM (n = 4). ∗p < 0.05, ∗∗p < 0.01 versus control groups, one-way ANOVA with Dunnet’s post hoc test. (D) Schematic representation of the in vitro BBB model. bEnd.3 cells were planted on microporous membrane of transwell inserts till confluence. After OGD/R treatment, the fluorescence dyes were added to upper compartment and the fluorescence intensity of lower compartment was measured 30 min later. (E and F) The transfer rate of dextran from upper compartment to lower compartment was examined to assess the endothelial monolayer permeability. Consistent diffused 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran during reoxygenation after 2 h OGD was limited by FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment. Data are expressed as mean ± SEM (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus ctrl group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus OGD + vehicle group, two-way ANOVA with Turkey’s post hoc test. (G) The endothelial monolayer permeability was further expressed as coefficient of diffusion (in centimeters per second). FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment significantly reduced the increased permeability of bEnd.3 monolayer to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran by exposure to OGD/R 3 h. Data are expressed as mean ± SEM (n = 5). ∗∗∗p < 0.001 versus ctrl group; #p < 0.05 versus OGD + vehicle (veh) group, one-way ANOVA with Sidak’s post hoc test. (H) PAK1 expression in bEnd.3 cells is downregulated by specific siPAK1, not siNC. Data are expressed as mean ± SEM (n = 3). ∗∗∗p < 0.001, Student’s t test. (I) Increased endothelial monolayer permeability of bEnd.3 cells to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran at OGD/R 3h was inhibited by siPAK1. Data are expressed as mean ± SEM (n = 5). ∗∗p < 0.01 versus ctrl group; #p < 0.05 versus OGD + siNC group, one-way ANOVA with Sidak’s post hoc test. See also Figure S2 .

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Inhibition, Western Blot, Control, In Vitro, Membrane, Fluorescence, Permeability, Diffusion-based Assay, Expressing

Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Recombinant, Plasmid Preparation, Lysis, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Cell Counting, Negative Control, Software

Journal: Frontiers in Molecular Biosciences

Article Title: SphK2/S1P Promotes Metastasis of Triple-Negative Breast Cancer Through the PAK1/LIMK1/Cofilin1 Signaling Pathway

doi: 10.3389/fmolb.2021.598218

Figure Lengend Snippet: Effects of IPA-3 on TNBC cell migration. (A) The influence of IPA-3 on TNBC cell viability was evaluated by a CCK-8 assay. (B) The level of phosphorylated PAK1 protein in TNBC cells treated with 5 μM IPA-3 for 24 h was measured by Western blot assay. (C,D) TNBC cell migration was examined after treatment with 5 μM IPA-3 by wound-healing and transwell assays. (E,F) SphK2-overexpressing TNBC cells were exposed to 5 μM IPA-3, and migration was evaluated. (G,H) TNBC cells treated with S1P were exposed to 5 μM IPA-3, and migration was evaluated. The results of each assay are representative of three independent experiments. The bars represent the mean ± SD of three replications of experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Immunoblotting was performed by incubation overnight at 4°C with the indicated primary antibodies (Cell Signaling Technology, Beverly, MA, United States except as noted): anti-PAK1, anti-p-PAK1, anti-Cofilin1, anti-p-Cofilin1, anti-LIMK1 (Abcam, Burlingame, CA, United States), anti-p-LIMK1 (Abcam, Burlingame, CA, United States), anti-SphK1 (Proteintech, Wuhan, China), and anti-SphK2 (Proteintech, Wuhan, China).

Techniques: Migration, CCK-8 Assay, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: SphK2/S1P Promotes Metastasis of Triple-Negative Breast Cancer Through the PAK1/LIMK1/Cofilin1 Signaling Pathway

doi: 10.3389/fmolb.2021.598218

Figure Lengend Snippet: The phosphorylation of PAK1, LIMK1, and Cofilin1 in different cell groups. Western blot assay on (A) the phosphorylation of PAK1, LIMK1, and Cofilin1 in TNBC cells transfected with SphK2 siRNA. (B) The phosphorylation of PAK1, LIMK1, and Cofilin1 in TNBC cells treated with ABC294640. (C) The phosphorylation of PAK1, LIMK1, and Cofilin1 in SphK2-overexpressing TNBC cells. (D) The phosphorylation of PAK1, LIMK1, and Cofilin1 in TNBC cells exposed to S1P. The results of each assay are representative of three independent experiments. The bars represent the mean ± SD of three replications of experiments. * p < 0.05, *** p < 0.001.

Article Snippet: Immunoblotting was performed by incubation overnight at 4°C with the indicated primary antibodies (Cell Signaling Technology, Beverly, MA, United States except as noted): anti-PAK1, anti-p-PAK1, anti-Cofilin1, anti-p-Cofilin1, anti-LIMK1 (Abcam, Burlingame, CA, United States), anti-p-LIMK1 (Abcam, Burlingame, CA, United States), anti-SphK1 (Proteintech, Wuhan, China), and anti-SphK2 (Proteintech, Wuhan, China).

Techniques: Western Blot, Transfection

Journal: Frontiers in Molecular Biosciences

Article Title: SphK2/S1P Promotes Metastasis of Triple-Negative Breast Cancer Through the PAK1/LIMK1/Cofilin1 Signaling Pathway

doi: 10.3389/fmolb.2021.598218

Figure Lengend Snippet: Inhibition of SphK2 activity reduces TNBC metastasis and the phosphorylation of PAK1, LIMK1, and Cofilin1 in vivo . (A) Representative images of the orthotopic tumors were obtained, and the volumes and weight of tumors were recorded. (B) Representative images of the lungs were obtained, and the metastatic nodules were counted. (C) Tumor apoptosis and proliferation were evaluated by TUNEL and Ki-67 staining, respectively. (D) The levels of SphK2 and phosphorylated PAK1, LIMK1, and Cofilin1 in orthotopic tumors and lung metastatic nodules were measured. The bars represent the mean ± SD of replications of experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.

Article Snippet: Immunoblotting was performed by incubation overnight at 4°C with the indicated primary antibodies (Cell Signaling Technology, Beverly, MA, United States except as noted): anti-PAK1, anti-p-PAK1, anti-Cofilin1, anti-p-Cofilin1, anti-LIMK1 (Abcam, Burlingame, CA, United States), anti-p-LIMK1 (Abcam, Burlingame, CA, United States), anti-SphK1 (Proteintech, Wuhan, China), and anti-SphK2 (Proteintech, Wuhan, China).

Techniques: Inhibition, Activity Assay, In Vivo, TUNEL Assay, Staining