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limk1 inhibitor limki330 32  (Tocris)
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Tocris limk1 inhibitor limki330 32
FIGURE 3 Arhgef6 regulates signaling to Cofilin via PAK2 and <t>LIMK1.</t> (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
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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

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

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

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

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

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