limk1 inhibitor limki330 32  (Tocris)

Journal: Journal of Leukocyte Biology

Article Title: Arhgef6 (alpha‐PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence

doi: 10.1002/jlb.1a1219-719r

Figure Lengend Snippet: FIGURE 3 Arhgef6 regulates signaling to Cofilin via PAK2 and LIMK1. (A) Western blots of total PAK2 and phospho-PAK2 (pPAK2) in wild-type (WT) and Arhgef6−/−(KO) CD4+ T cells with quantification (right). Total PAK2: Arhgef6−/−T cells normalized to WT, pPAK2: ratio of pPAK2/PAK2 for Arhgef6−/−T cells normalized to WT ratio. (B, C) Cells as in A, but for phospho-LIMK1 (pLIMK1) (B) and phospho-Cofilin (pCofilin) (C) (n = 3 experiments with one mouse of each genotype per experiment per Western blot). ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s t-test. Values are mean ± SEM

Article Snippet: In some experiments the cells were additionally treated by adding LIMK1 inhibitor LIMKi330–32 (10 μM) (4745, Tocris,Wiesbaden, Germany) for 4 h before harvesting or with ethanol alone as a vehicle control at 0.05%.

Techniques: Western Blot

Journal: Journal of Leukocyte Biology

Article Title: Arhgef6 (alpha‐PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence

doi: 10.1002/jlb.1a1219-719r

Figure Lengend Snippet: FIGURE 7 LIMK1 inhibition increases wild-type (WT) T cell migration speed. (A) Total internal reflection fluorescence (TIRF) images from Supporting Information Videos S5 to S8 (t = 10 s each) of WT and Arhgef6−/−CD4+ T cells treated with control ethanol vehicle or LIMK1 inhibitor, LIMKi3 (10 𝜇M for 4 h), stained with CellBrite live cell membrane dye and migrating on ICAM-1 display increased lamellipodia size for WT cells. Scale bar, 5 𝜇m. (B) LIMK1 inhibition in WT T cells causes increased lamellipodial width and area but has no effect on Arhgef6−/−T cells. Quantifi- cation of lamellipodial length (left), width (center), and area (right) for WT and Arhgef6−/−T cells either untreated vehicle control (ctrl) or treated with LIMKi3 as shown in (A). Bars = mean ± SEM, (n = 3 experiments with 30 cells each). ns = not significant, ****P < 0.0001, by 2-way ANOVA followed by Bonferroni’s post hoc test. (C) Representative track plots for WT and Arhgef6−/−T cells migrating on ICAM-1 either untreated (ctrl) or treated with LIMKi3. (D) Quantification of velocity, displacement and straightness for the samples shown in (c). Mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, by Student’s t-test

Article Snippet: In some experiments the cells were additionally treated by adding LIMK1 inhibitor LIMKi330–32 (10 μM) (4745, Tocris,Wiesbaden, Germany) for 4 h before harvesting or with ethanol alone as a vehicle control at 0.05%.

Techniques: Inhibition, Migration, Fluorescence, Control, Staining, Membrane

Journal: Journal of Leukocyte Biology

Article Title: Arhgef6 (alpha‐PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence

doi: 10.1002/jlb.1a1219-719r

Figure Lengend Snippet: FIGURE 8 Schematic representation of Arhgef6-controlled signaling pathways. In wild-type (WT) cells, Arhgef6 and Arhgef7, RhoGEFs for Rac1 and Cdc42, repress signaling to actin reorganization and restrict lamellipodial formation to limit cell speed and maintain rela- tive straightness. In T cells lacking Arhgef6, cells migrate faster and turn more. Cdc42 is mislo- calized to the ICAM1-coated migration surface and Rac1 is overactivated. Moreover, PAK2, LIMK1, and Cofilin are all hypophosphorylated meaning that Cofilin, which promotes actin severing and polymerization, is overactivated. The mechanisms for Rac1 activation of lamel- lipodial extension are not characterized here but may include hyperativation of WAVE and Arp2/3, both required for lamellipodia exten- sion. Arhgef7 expression is increased, likely due to its taking the place of Arhgef6 in the PIX-GIT complex, but it cannot compensate fully for the absence of Arhgef6 as the immune cell-specific Arhgef6 may be required for targeting the complex to T cell specific receptors

Article Snippet: In some experiments the cells were additionally treated by adding LIMK1 inhibitor LIMKi330–32 (10 μM) (4745, Tocris,Wiesbaden, Germany) for 4 h before harvesting or with ethanol alone as a vehicle control at 0.05%.

Techniques: Protein-Protein interactions, Migration, Activation Assay, Expressing

Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: a , Schematic illustration of SEMA3A signaling. Upon SEMA3A binding to NRP1, NRP1 forms a holoreceptor complex with a plexin, which acts as the signal-transducing unit. Through a signaling cascade, LIMK1 is activated and catalyzes the phosphorylation of cofilin-1. Cofilin-1 is an actin depolymerization factor, which is de-activated upon phosphorylation at its serine 3 (S3). Thus, less actin is depolymerized, resulting in a less dynamic actin network and, subsequently, fewer fenestrae. b , Western blots of mouse LSEC protein lysates ( n = 5 independent LSEC isolations). LSECs were pretreated with either DMSO or LIMKi 3, a LIMK1 inhibitor, and then treated with either SEMA3A-Fc or IgG2a-Fc. For the analysis, cofilin-1 and p-S3-cofilin-1 were normalized to GAPDH and then put into relation of each other (p-S3-cofilin-1 to cofilin-1). c , Representative SEM images of mouse LSECs pretreated with either DMSO or LIMKi 3 and then treated with either SEMA3A-Fc or IgG2a-Fc. The fenestrae were colorized with a digital charcoal pencil for better visualization. Scale bar, 1 µm. Brightness and contrast have been adjusted to enhance visibility in b , c . d – f , Analyses of fenestrae frequency ( d ) and diameter ( e ) as well as porosity ( f ) of mouse LSECs pretreated with LIMKi 3 or DMSO and subsequently treated with SEMA3A-Fc or IgG2a-Fc, as indicated. For each condition, ten images (taken from different LSECs) were analyzed ( n = 5 LSEC isolations). For statistical analysis, a one-way ANOVA with multiple comparisons (Tukey’s post hoc test) was performed in b , d – f . In all graphs, data points and mean ± s.e.m. are presented.

Article Snippet: After addition of the antibodies, the cells were incubated at 37 °C and 5% CO 2 for 1 h. If LSECs were to be pretreated with the LIMK1 inhibitor LIMKi 3 (Tocris, 4745), they were allowed to grow 4 h and then incubated with LIMKi 3 for 1 h at 37 °C and 5% CO 2 .

Techniques: Binding Assay, Phospho-proteomics, Western Blot

Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: Left side: in the setting of low physiological SEMA3A levels (as is the case at low concentrations of saturated fatty acids and normal BW without T2D), active cofilin-1 and normal F-actin cytoskeleton dynamics contribute to maintain a high frequency of fenestrae in LSECs. LSEC porosity facilitates bidirectional exchange of lipids between bloodstream and hepatocytes, such as the release of VLDL particles from hepatocytes into the blood circulation. Right side: in the setting of high SEMA3A levels (as is the case at high concentrations of FFAs and in DIO with or without T2D), the angiocrine signal SEMA3A acts via NRP1 on LSECs to activate multiple STKs, including LIMK1, which phosphorylates cofilin-1 to reduce F-actin cytoskeleton dynamics and fenestrae frequency as well as LSEC porosity. The reduced LSEC porosity lowers VLDL export from the hepatocytes into the blood and might contribute to lipid retention and macrovesicular steatosis in the hepatocytes. The resulting hepatic steatosis is an early event in MASLD that can subsequently (in concert with hepatic stellate cells; HSCs) progress to severe hepatic and cardiometabolic diseases. The figure was created with BioRender.com .

Article Snippet: After addition of the antibodies, the cells were incubated at 37 °C and 5% CO 2 for 1 h. If LSECs were to be pretreated with the LIMK1 inhibitor LIMKi 3 (Tocris, 4745), they were allowed to grow 4 h and then incubated with LIMKi 3 for 1 h at 37 °C and 5% CO 2 .

Techniques:

Journal: FASEB bioAdvances

Article Title: Cyclosporine A inhibits MRTF‐SRF signaling through Na + /K + ATPase inhibition and actin remodeling

doi: 10.1096/fba.2019-00027

Figure Lengend Snippet: Cofilin is involved in cyclosporine A (CsA)‐induced actin reorganization through its phosphorylation site. A, Fluorescence images of Lilly Laboratories Porcine Kidney‐1 actin cytoskeleton labeled with TRITC‐Phalloidin. Scale bar 10 µm. B, Quantification of red fluorescence‐positive area. Mean ± SEM. One‐way ANOVA plus Tukey's post‐test (* P < .05, *** P < .001) (n = 4). Drug condition: (a) Vehicle (b) 100 µg/mL S3R (c) 10 µmol/L LIMKi3 (d) 5 µmol/L CsA (e) S3R + CsA (f) LIMKi3 + CsA. Exposure time: 24 h (n = 4)

Article Snippet: LIMK1/2 inhibitor LIMKi3 (#4745) was provided by Tocris.

Techniques: Phospho-proteomics, Fluorescence, Labeling

Journal: FASEB bioAdvances

Article Title: Cyclosporine A inhibits MRTF‐SRF signaling through Na + /K + ATPase inhibition and actin remodeling

doi: 10.1096/fba.2019-00027

Figure Lengend Snippet: Cofilin is involved in cyclosporine A (CsA)‐induced inhibition of myocardin‐related transcription factors‐serum response factor (SRF) transcription activity. SRF transcription activity was measured by luciferase gene reporter assay in Lilly Laboratories Porcine Kidney‐1 SRE exposed 24 h to: (A) S3‐R 100 µg/mL; S3R + CsA. (n = 6); (B) LIMKi3 10 µmol/L; LIMKi3 + CsA. (n = 7). Mean ± SEM. One‐sample t test for vs control comparison, One‐way ANOVA plus Tukey's post‐test for multiple condition comparison (* P < .01, *** P < .001)

Article Snippet: LIMK1/2 inhibitor LIMKi3 (#4745) was provided by Tocris.

Techniques: Inhibition, Activity Assay, Luciferase, Reporter Assay, Control, Comparison