Journal: Advanced Science

Article Title: Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

doi: 10.1002/advs.202502907

Figure Lengend Snippet: Identification of PKN2 and MOB4 as genes that control the coordination of collective cell migration. A) Fields of displacement vectors are obtained by Particle Image Velocimetry (PIV) of the wound healing movie. The vector field is superimposed on the phase contrast image of the MCF10A monolayer. Definition of migration parameters extracted from vector fields. Scale bar: 100 µm. B) Speed and order parameter are plotted as heat maps across time and space. This heat map representation shows the monolayer front‐facing the wound on top and time 0 on the left. For one KO clone of each candidate gene, averaged heat maps of eight movies from two independent experiments are plotted. Each independent experiment gave similar results. The dashed line represents the time 8 h. C) Local speed and order are averaged at 8 h after wounding from the 12 fields of view of the three biological repeats. Mean ± SEM, two‐way ANOVA. D) Velocity correlation length calculated from 11 fields of view from the three biological repeats is plotted as a function of time. Areas under the curve are compared by one‐way ANOVA, mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ns non‐significant.

Article Snippet: The following commercial antibodies were used: DLG5 (ThermoFisher Scientific #A302‐302A‐M), PKN2 (Proteintech #14608‐1‐AP), AMOTL2 (Proteintech #23351‐1‐AP), TP53BP2 (Proteintech #26550‐1‐AP), MOB4 (Proteintech #15886‐1‐AP), E‐cadherin (clone DECMA‐1, Sigma–Aldrich #MABT26), CYFIP2 (Sigma–Aldrich #SAB2701081), NCKAP1 (Bethyl Laboratories, A305‐178A), Abi1 (ThermoFischer Scientific #PA5‐78705), Cortactin (clone 4F11, Merck 05‐180‐I), YAP1 (clone 63.7, SantaCruz Biotechnology #sc‐101199), phospho‐YAP1 (Ser127) (Proteintech #80694‐2‐RR, PtgLab), phospho‐YAP1 (Ser397) (Proteintech #29018‐1‐AP), GFP (clones 13.1 and 7.1, Sigma–Aldrich #11814460001), Tubulin (clone DM1A, Sigma–Aldrich #T9026), p150 Glued /DCTN1 (BD Biosciences #BD610474), GAPDH (ThermoFisher Scientific #AM4300).

Techniques: Control, Migration, Plasmid Preparation

Journal: Advanced Science

Article Title: Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

doi: 10.1002/advs.202502907

Figure Lengend Snippet: PKN2 and MOB4 differentially localize to the front and lateral edges of collectively migrating cells. A) Stable MCF10A lines expressing GFP‐PKN2 or GFP‐MOB4 are imaged by confocal microscopy. Filamentous actin is stained with phalloidin, and nuclei with DAPI. Angle distribution of all cell junctions labeled by MOB4 or PKN2. In total, more than 100 junctions per cell line are assessed from at least 10 images in three independent experiments. B) PKN2 and MOB4 immunofluorescence in MCF10A monolayers at time 0 or 8 h after wounding. Filamentous actin is stained with phalloidin, and nuclei with DAPI. Angle distribution of all cell junctions labeled with MOB4 or PKN2. More than 50 junctions for each staining are assessed from at least 10 images in two independent experiments. Confocal microscopy. Scale bars 20 µm.

Article Snippet: The following commercial antibodies were used: DLG5 (ThermoFisher Scientific #A302‐302A‐M), PKN2 (Proteintech #14608‐1‐AP), AMOTL2 (Proteintech #23351‐1‐AP), TP53BP2 (Proteintech #26550‐1‐AP), MOB4 (Proteintech #15886‐1‐AP), E‐cadherin (clone DECMA‐1, Sigma–Aldrich #MABT26), CYFIP2 (Sigma–Aldrich #SAB2701081), NCKAP1 (Bethyl Laboratories, A305‐178A), Abi1 (ThermoFischer Scientific #PA5‐78705), Cortactin (clone 4F11, Merck 05‐180‐I), YAP1 (clone 63.7, SantaCruz Biotechnology #sc‐101199), phospho‐YAP1 (Ser127) (Proteintech #80694‐2‐RR, PtgLab), phospho‐YAP1 (Ser397) (Proteintech #29018‐1‐AP), GFP (clones 13.1 and 7.1, Sigma–Aldrich #11814460001), Tubulin (clone DM1A, Sigma–Aldrich #T9026), p150 Glued /DCTN1 (BD Biosciences #BD610474), GAPDH (ThermoFisher Scientific #AM4300).

Techniques: Expressing, Confocal Microscopy, Staining, Labeling, Immunofluorescence

Journal: Advanced Science

Article Title: Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

doi: 10.1002/advs.202502907

Figure Lengend Snippet: PKN2 KO cells display weakened adherens junctions. A) E‐cadherin immunofluorescence of epithelial islets of PKN2, MOB4 KO, and control cells. Filamentous actin is stained with phalloidin, and nuclei with DAPI. Confocal microscopy. Scale bar 20 µm. Black‐and‐white inserts show E‐cadherin staining in the magnified regions. Three independent experiments gave similar results. Intensity profiles taken perpendicularly to cell–cell junctions were averaged for a total of n = 60 junctions from 9 images per condition. Mean ± SEM. Areas under the curve, one‐way ANOVA. B) Western blots of E‐cadherin, PKN2, MOB4, and tubulin as a loading control. C) Islets of PKN2 KO, MOB4 KO, and parental cells are stained with antibodies to cortactin, phalloidin, and DAPI. Confocal microscopy. Scale bar 20 µm. Black‐and‐white inserts show cortactin staining in the magnified regions. Intensity profiles of cortactin taken perpendicularly to cell–cell junctions were averaged for a total of n = 20 junctions measured from 3 images per condition. Mean ± SEM. D) Measurements of the rupture force of E‐cadherin‐coated beads in PKN2 KO and MCF10A parental cells. The control shown refers to force measurement when the beads are not coated. The setup used consists of a chamber composed of a U‐shaped PDMS structure between two glass coverslips, where cells are seeded and manipulated. The liquid reservoir, when placed below the plane of the cells, allows aspirating beads. When the micromanipulator is used to displace the bead, the micropipette bends because of bead attachment to the cell until the bonds rupture. The force of the E‐cadherin‐mediated interaction is calculated from the micropipette deflection, knowing the measured elasticity of the micropipette. Non‐paired t‐test. * p<0.05, ns non‐significant.

Article Snippet: The following commercial antibodies were used: DLG5 (ThermoFisher Scientific #A302‐302A‐M), PKN2 (Proteintech #14608‐1‐AP), AMOTL2 (Proteintech #23351‐1‐AP), TP53BP2 (Proteintech #26550‐1‐AP), MOB4 (Proteintech #15886‐1‐AP), E‐cadherin (clone DECMA‐1, Sigma–Aldrich #MABT26), CYFIP2 (Sigma–Aldrich #SAB2701081), NCKAP1 (Bethyl Laboratories, A305‐178A), Abi1 (ThermoFischer Scientific #PA5‐78705), Cortactin (clone 4F11, Merck 05‐180‐I), YAP1 (clone 63.7, SantaCruz Biotechnology #sc‐101199), phospho‐YAP1 (Ser127) (Proteintech #80694‐2‐RR, PtgLab), phospho‐YAP1 (Ser397) (Proteintech #29018‐1‐AP), GFP (clones 13.1 and 7.1, Sigma–Aldrich #11814460001), Tubulin (clone DM1A, Sigma–Aldrich #T9026), p150 Glued /DCTN1 (BD Biosciences #BD610474), GAPDH (ThermoFisher Scientific #AM4300).

Techniques: Immunofluorescence, Control, Staining, Confocal Microscopy, Western Blot

Journal: Advanced Science

Article Title: Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

doi: 10.1002/advs.202502907

Figure Lengend Snippet: EDTA treatment, which weakens adherens junctions, phenocopies PKN2 KO cells. A) Wound healing of PKN2 KO cells or parental cells treated with 1 mM EDTA to destabilize cell–cell junctions or 1 mM EDTA with 1 mM CaCl 2 to neutralize EDTA. EDTA‐mediated destabilization of junctions phenocopies the PKN2 KO phenotype. Scale bar 100 µm. B) Velocity correlation length, calculated from 12 movies from two independent experiments, is plotted as a function of time. Areas under the curve are compared by one‐way ANOVA, mean ± SEM, **** p <0.0001, ns non‐significant. C) Heat maps of speed and order parameters are extracted from PIV analyses of 12 movies from two independent experiments per condition. Each independent experiment gave similar results.

Article Snippet: The following commercial antibodies were used: DLG5 (ThermoFisher Scientific #A302‐302A‐M), PKN2 (Proteintech #14608‐1‐AP), AMOTL2 (Proteintech #23351‐1‐AP), TP53BP2 (Proteintech #26550‐1‐AP), MOB4 (Proteintech #15886‐1‐AP), E‐cadherin (clone DECMA‐1, Sigma–Aldrich #MABT26), CYFIP2 (Sigma–Aldrich #SAB2701081), NCKAP1 (Bethyl Laboratories, A305‐178A), Abi1 (ThermoFischer Scientific #PA5‐78705), Cortactin (clone 4F11, Merck 05‐180‐I), YAP1 (clone 63.7, SantaCruz Biotechnology #sc‐101199), phospho‐YAP1 (Ser127) (Proteintech #80694‐2‐RR, PtgLab), phospho‐YAP1 (Ser397) (Proteintech #29018‐1‐AP), GFP (clones 13.1 and 7.1, Sigma–Aldrich #11814460001), Tubulin (clone DM1A, Sigma–Aldrich #T9026), p150 Glued /DCTN1 (BD Biosciences #BD610474), GAPDH (ThermoFisher Scientific #AM4300).

Techniques:

Journal: Advanced Science

Article Title: Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration

doi: 10.1002/advs.202502907

Figure Lengend Snippet: Working model of the roles of PKN2 and MOB4 in the control of collective cell migration. PKN2 localizes at lateral junctions and maintains cohesiveness of the cell monolayer through the maintenance of adherens junctions, whereas MOB4 localizes at the front edge and indicates the cell where to migrate through YAP1 activation. In addition, MOB4 restricts the territory of collectively migrating cells to the first rows of cells facing the wound.

Article Snippet: The following commercial antibodies were used: DLG5 (ThermoFisher Scientific #A302‐302A‐M), PKN2 (Proteintech #14608‐1‐AP), AMOTL2 (Proteintech #23351‐1‐AP), TP53BP2 (Proteintech #26550‐1‐AP), MOB4 (Proteintech #15886‐1‐AP), E‐cadherin (clone DECMA‐1, Sigma–Aldrich #MABT26), CYFIP2 (Sigma–Aldrich #SAB2701081), NCKAP1 (Bethyl Laboratories, A305‐178A), Abi1 (ThermoFischer Scientific #PA5‐78705), Cortactin (clone 4F11, Merck 05‐180‐I), YAP1 (clone 63.7, SantaCruz Biotechnology #sc‐101199), phospho‐YAP1 (Ser127) (Proteintech #80694‐2‐RR, PtgLab), phospho‐YAP1 (Ser397) (Proteintech #29018‐1‐AP), GFP (clones 13.1 and 7.1, Sigma–Aldrich #11814460001), Tubulin (clone DM1A, Sigma–Aldrich #T9026), p150 Glued /DCTN1 (BD Biosciences #BD610474), GAPDH (ThermoFisher Scientific #AM4300).

Techniques: Control, Migration, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

doi: 10.1074/jbc.m113.454082

Figure Lengend Snippet: FIGURE 1. Pak1 mediates MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. A, equal amounts of protein from quiescent and MCP1-treated (50 ng/ml) HASMCs were analyzed by Western blotting for pPak1 levels using its phosphospecific antibodies, and the blot was reprobed for total Pak1 levels for normalization. To measure Pak1 activity, equal amounts of protein from the control and each treatment were immunoprecipitated with anti-Pak1 antibodies, and the immunocomplexes were assayed for its kinase activity using MBP and [-32P]ATP as substrates. B, quiescent and MCP1-treated (50 ng/ml) HASMCs were stained with phalloidin for F-actin stress fiber formation. C, upper panel, HASMCs were transfected with non-targeted control or SMARTpool Pak1 siRNA (100 nM). Two days after transfection, cell extracts were prepared, equal amounts of protein from each condition were analyzed by Western blotting for Pak1 levels using its specific antibodies, and the blot was reprobed for -tubulin levels to show the effect of the siRNA on its target and off-targetmoleculelevels.Lowerpanel,HASMCsthatweretransfectedwithnon-targetedcontrolorSMARTpoolPak1siRNA(100nM)andquiescedweretreated with and without MCP1 (50 ng/ml) for 8 h and stained with phalloidin for F-actin stress fiber formation. D and E, HASMCs that were transfected with non-targeted control or SMARTpool Pak1 siRNA (100 nM) and quiesced were treated with and without MCP1 (50 ng/ml) for 24 h and 8 h, and DNA synthesis (D) and cell migration (E) were measured, respectively. F, HASMCs that were transfected with non-targeted control or siGENOME Pak1 siRNA (100 nM) and quiesced were treated with and without MCP1 (50 ng/ml) for 8 h. Cell extracts were prepared, and equal amounts of protein from control and each treatment were analyzedforPak1activityasdescribedinA.EqualamountsofproteinfromthesamecellextractswerealsoanalyzedbyWesternblottingforPak1and-tubulin levels to show the effect of the siRNA on its target and off-target molecule levels. SiGsiRNA, siGENOME siRNA. G and H, all the conditions were the same as in F, except that after quiescence, cells were treated with and without MCP1 (50 ng/ml) for 24 h and 8 h, and DNA synthesis (G) and migration (H) were measured, respectively. DNA synthesis was measured by [3H]thymidine incorporation, and cell migration was measured by the Boyden chamber method. *, p 0.01 versus control siRNA; **, p 0.01 versus control siRNA MCP1.

Article Snippet: Anti-pPak1 (catalog no. 2601) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Migration, Western Blot, Activity Assay, Control, Immunoprecipitation, Staining, Transfection, DNA Synthesis

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

doi: 10.1074/jbc.m113.454082

Figure Lengend Snippet: FIGURE 2. CDK4/6 mediate MCP1-induced Pak1 activation in HASMCs. A, quiescent HASMCs were treated with and without MCP1 (50 ng/ml) for the indicated time periods, and cell extracts were prepared. Equal amounts of protein from the control and each treatment were analyzed by immunocomplex kinase assay for CDK4/6 activities using truncated recombinant retinoblastoma protein and [-32P]ATP as substrates. B, after transfection with non-targeted control or SMARTpool CDK6 or CDK4 siRNA (100 nM) and quiescence, HASMCs were treated with and without MCP1 (50 ng/ml) for 8 h, and cell extracts were prepared. Equal amounts of proteins from the control and each treatment were analyzed by Western blotting for pPak1 levels using its phosphospecific antibodies, and the blot was reprobed for Pak1, CDK6, CDK4, or -tubulin levels for normalization or to show the effects of the siRNAs on their target and off-target molecule levels. Equal amounts of protein from control and each treatment were also assayed for Pak1 activity by immunocomplex kinase assay using MBP and [-32P]ATP as substrates. C, conditions were the same as in B, except that after treatment with and without MCP1, cells were stained with phalloidin for F-actin stress fiber formation. D and E, after transfections with non-targeted control or SMARTpool CDK6 or CDK4 siRNA and quiescence, cells weretreatedwithandwithoutMCP1(50ng/ml)for24hand8h,andDNAsynthesis(D)andcellmigration(E)weremeasured,respectively.F,HASMCsthatwere transfected with non-targeted control or SMARTpool CDK6 or CDK4 siRNA and quiesced were treated with and without MCP1 (50 ng/ml) for 8 h. Cell extracts were prepared, and equal amounts of protein from the control and each treatment were analyzed by immunocomplex kinase assay for CDK6 and CDK4 activities as described in A. Cell extracts containing an equal amount of protein from the control and each treatment were also analyzed by Western blotting for CDK6 and CDK4 levels to show the effect of their siRNAs on their total levels. G and H, quiescent cells were treated with and without MCP1 (50 ng/ml) in the presence and absence of fascaplysin (5 M) for 8 h, and either cell extracts were prepared and analyzed for CDK4 and Pak1 activities as described in A and B, respectively, or stained with phalloidin for F-actin stress fiber formation as described in C. *, p 0.01 versus control siRNA; **, p 0.01 versus control siRNA MCP1.

Article Snippet: Anti-pPak1 (catalog no. 2601) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Control, Kinase Assay, Recombinant, Transfection, Western Blot, Activity Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

doi: 10.1074/jbc.m113.454082

Figure Lengend Snippet: FIGURE 3. NFATc1 mediates MCP1-induced cyclin D1 expression, CDK4/6 activities, and Pak1 activation in HASMCs. A, quiescent HASMCs were treated with and without MCP1 (50 ng/ml) for the indicated time periods, and cell extracts were prepared. Equal amounts of protein from the control and each treatment were analyzed by Western blotting for cyclin D1 levels using its specific antibodies, and the blot was reprobed for -tubulin levels for normalization. B, HASMCs that were transfected with non-targeted control or SMARTpool NFATc1 or cyclin D1 siRNA (100 nM) and quiesced were treated with and without MCP1 (50 ng/ml) for 8 h, and cell extracts were prepared. Cell extracts consisting of equal amounts of protein from the control and each treatment were analyzed for cyclin D1 and pPak1 levels by Western blotting using their normal or phosphospecific antibodies or assayed for CDK4/6 and Pak1 activities by immunocomplex kinase assays using truncated recombinant retinoblastoma protein and [-32P]ATP as substrates for CDK4/6 activities and MBP and [-32P]ATPassubstratesforPak1activity.ThecyclinD1blotwasreprobedsequentiallyforNFATc1and-tubulinlevelstoshowtheeffectsofthesiRNAsontheir target and off-target molecule levels. C, all conditions were the same as in B, except that after treatment with and without MCP1, cells were stained with phalloidin for F-actin stress fiber formation. D and E, after transfections with non-targeted control, SMARTpool NFATc1, or cyclin D1 siRNA and quiescence, cells were treated with and without MCP1 (50 ng/ml) for 8 h and 24 h, and cell migration (D and E, right panels) and DNA synthesis (left panels) were measured, respectively. F, HASMCs that were transfected with non-targeted control or siGENOME cyclin D1 or CDK6 siRNA (100 nM) and quiesced were treated with and without MCP1 (50 ng/ml) for 8 h. Cell extracts were prepared, and equal amounts of protein from the control and each treatment were analyzed for CDK4 and Pak1activitiesasdescribedinB.EqualamountsofproteinfromthesamecellextractswerealsoanalyzedbyWesternblottingforcyclinD1,CDK6,and-tubulin levels to show the effects of the siRNAs on their target and off-target molecule levels. SiGsiRNA, siGENOME siRNA. G and H, all conditions were the same as in F, except that after quiescence, cells were treated with and without MCP1 (50 ng/ml) for 24 h and 8 h, and DNA synthesis and migration were measured as described in D. The bar graph in each panel represents the quantitative analysis of three independent experiments. *, p 0.01 versus control siRNA; **, p 0.01 versus control siRNA MCP1.

Article Snippet: Anti-pPak1 (catalog no. 2601) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activation Assay, Control, Western Blot, Transfection, Recombinant, Staining, Migration, DNA Synthesis

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

doi: 10.1074/jbc.m113.454082

Figure Lengend Snippet: FIGURE5.Rac1actsupstreamofMCP1-inducedactivationofNFATc1-cyclinD1-CDK6-CDK4-Pak1signalinginthemodulationofHASMCF-actinstress fiber formation, migration, and proliferation. A, quiescent HASMCs were treated with and without MCP1 (50 ng/ml) for the indicated time periods, and cell extracts were prepared. Equal amounts of protein from the control and each treatment were analyzed by pull-down assays for Rac1 and RhoA activation using GST-Pak1 and GST-Rhotekin beads, respectively. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for total Rac1 and RhoA levels using their specific antibodies. B–D, HASMCs that were transduced with Ad-GFP or Ad-dnRac1 (109 plaque-forming units) and quiesced were subjected to MCP1 (50 ng/ml)-induced F-actin stress fiber formation (B), DNA synthesis (C), or migration (D). E, all conditions were the same as in B, except that after treatment with and without MCP1, cytoplasmic and nuclear extracts were prepared, and equal amounts of protein from each cell fraction were analyzed by Western blotting for NFATc1 levels using its specific antibodies. The blot was reprobed sequentially for MEK1, p53, and -tubulin levels to show the purity of the cytoplasmic (CF) and nuclear (NF) extracts. F, all conditions were the same as in B, except that after treatment with and without MCP1, cell extracts were prepared, and equal amounts of protein from the control and each treatment were analyzed by either Western blotting for cyclin D1, pPak1, Pak1, Rac1, GFP, and -tubulin levels using their specific antibodies or immunocomplex kinase assays for CDK6/4 and Pak1 activities using truncated recombinant retinoblastoma protein and MBP along with [-32P]ATP as substrates, respectively. *, p 0.01 versus Ad-GFP; **, p 0.01 versus Ad-GFP MCP1.

Article Snippet: Anti-pPak1 (catalog no. 2601) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Migration, Control, Activation Assay, Western Blot, Transduction, DNA Synthesis, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

doi: 10.1074/jbc.m113.454082

Figure Lengend Snippet: FIGURE 7. SM-specific knockdown of NFTAc1 prevents MCP1-induced cyclin D1 expression, CDK6/4 activities, and Pak1 activation in VSMCs in vitro and balloon injury-induced neointima formation in vivo. A, upper panel, DNA was isolated from WT and SM22Cre:NFATc1fl/fl mice tail biopsies, and genotypingwasperformedasdescribedunder“MaterialsandMethods.”Lowerpanel,anequalamountofproteinfromWTandSM22Cre:NFATc1fl/flVSMCswas analyzed by Western blotting for NFATc1 levels using its specific antibodies. B, quiescent VSMCs from WT and SM22Cre:NFATc1fl/fl mice were treated with and without MCP1 (50 ng/ml) for 8 h, and cell extracts were prepared. Equal amounts of protein from the control and each treatment were analyzed for GTP-Rac1, cyclin D1, pPak1, Pak1, and -tubulin levels or CDK6/4 and Pak1 activities as described in Figs. 5A and 6B. C and D, quiescent VSMCs from WT and SM22Cre: NFATc1fl/fl mice were treated with and without MCP1 (50 ng/ml) for 24 h and 8 h, and DNA synthesis (C) and cell migration (D) were measured, respectively. E, WT and SM22Cre:NFATc1fl/fl mice were subjected to guide wire injury, and 3 weeks post-guide wire injury, the arteries were isolated and fixed. Cryosections were made and stained for MCP1, pPak1, and SMC-actin using their specific antibodies as described under “Materials and Methods.” F, all conditions were the same as in E, except that sections were stained with H&E, and the I/M ratios were calculated. The left panel shows the representative pictures of balloon-injured commoncarotidarterycross-sectionsthatwerestainedwithH&E.ThebargraphintherightpanelshowsthequantitativeanalysisoftheI/Mratiosoftheinjured common carotid arteries of six animals. *, p 0.05 versus WT guide wire injury.

Article Snippet: Anti-pPak1 (catalog no. 2601) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Knockdown, Expressing, Activation Assay, In Vitro, In Vivo, Isolation, Western Blot, Control, DNA Synthesis, Migration, Staining

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 1. ADAM15-dependent mechano-induced downregulation of lncRNA H19 is affected by inhibition of p21-activated kinases (PAKs). (A, B) Synovial fibroblasts either silenced with an ADAM15 siRNA or a non-silencing negative siRNA were mechanically strained (1 Hz and 15% elongation) for 1–9 h and H19 levels determined by RT-qPCR. (A) Fold changes of H19 in ADAM15-expressing versus non-expressing cells were calculated using the 2−ΔΔCt. Shown is the mean ± SD of 6 different RASFs. (B) time course of GAPDH- normalized Ct values for H19 upon mechanical strain, showing increase of Ct values (i.e. lower H19 amounts) in ADAM15 expressing cells (black dots) compared to ADAM15 non-expressing cells (open circle) in one representative RASF cell line. (C) RT-qPCR for H19 of RASFs stimulated for 1 and 3 h in the presence of either DMEM medium, or inhibitors for JNK (SP600125), Src family kinases (dasatinib), CAMKII (KN- 93), calmodulin (TFP) and PAKs (IPA-3). **p < 0.005, ***p < 0.0005, Student’s t-test, comparing ADAM15- expressing versus non-expressing cells or inhibitor versus DMEM.

Article Snippet: Samples were applied in loading buffer (6x stock solution: 375 mM Tris-HCl, 9% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), 9% β-mercaptoethanol (v/v)), separated by 10% SDS/PAGE, Scientific Reports | (2025) 15:9814 12| https://doi.org/10.1038/s41598-025-94012-2 transferred to nitrocellulose filter, and blocked with 5% milk powder (Roth) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, 0,1% Tween20, pH 7.4) for 30 min at room temperature and incubated with antibodies diluted in 1% milk powder/PBST against phospho-PAK2 (Ser20) (Cell Signaling Technology, #2607, dilution 1:1000), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:1000), Nck1 (Cell Signaling Technology, #2319, 1:1000), PAK2 (R&D Systems, #MAB6849; 1:1000), ADAM15 (R&D Systems, #AF935, 1:1000), OB-Cadherin/Cadherin-11 (Santa Cruz Biotechnology, #sc-365867; 1:1000), N-cadherin (R&D Systems, #AF6426; 1:1000), GAPDH (Santa Cruz Biotechnology, #sc-365062; 1:10.000) overnight at 4 °C and developed using appropriate horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, horse anti-mouse IgG #7076, horse anti-rabbit IgG #7074 (1:2500 each), donkey antiGoat IgG (Thermo Fisher #A15999; 1:2500).

Techniques: Inhibition, Quantitative RT-PCR, Expressing

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 2. ADAM15- and N-cadherin (NCAD) dependent activation of PAK2 upon mechanical strain. (A–D) Immunoblots for pPAK2. (A) of RASFs seeded at different cell densities and mechanically strained for 30 and 60 min. (B) from strained RASFs with PAK2 silencing (si) and treated with a negative control siRNA (neg). (C, D) immunoblots for pPAK2, ADAM15 and NCAD from RASFs treated with ADAM15 and NCAD siRNA; right panels, showing the mean ± SD of densitometric analysis of 6 different RASF cell lines. ***p < 0.0005, Student’s t-test. GAPDH served as a loading control.

Article Snippet: Samples were applied in loading buffer (6x stock solution: 375 mM Tris-HCl, 9% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), 9% β-mercaptoethanol (v/v)), separated by 10% SDS/PAGE, Scientific Reports | (2025) 15:9814 12| https://doi.org/10.1038/s41598-025-94012-2 transferred to nitrocellulose filter, and blocked with 5% milk powder (Roth) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, 0,1% Tween20, pH 7.4) for 30 min at room temperature and incubated with antibodies diluted in 1% milk powder/PBST against phospho-PAK2 (Ser20) (Cell Signaling Technology, #2607, dilution 1:1000), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:1000), Nck1 (Cell Signaling Technology, #2319, 1:1000), PAK2 (R&D Systems, #MAB6849; 1:1000), ADAM15 (R&D Systems, #AF935, 1:1000), OB-Cadherin/Cadherin-11 (Santa Cruz Biotechnology, #sc-365867; 1:1000), N-cadherin (R&D Systems, #AF6426; 1:1000), GAPDH (Santa Cruz Biotechnology, #sc-365062; 1:10.000) overnight at 4 °C and developed using appropriate horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, horse anti-mouse IgG #7076, horse anti-rabbit IgG #7074 (1:2500 each), donkey antiGoat IgG (Thermo Fisher #A15999; 1:2500).

Techniques: Activation Assay, Western Blot, Negative Control, Control

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 4. Mechano-induced recruitment of PAK2 together with Nck to the cell membrane. (A, B) RASFs grown on Bioflex plates and either unstimulated or strained for 30 min were fluorescently stained for (A) NCAD and Nck and (B) NCAD and PAK2. Adjacent to images: quantification of the pixel density of NCAD /Nck or NCAD /PAK2 along the white arrow. (C) double staining of NCAD and PAK2 in RASFs with prior silencing of Nck, right, pixel density along arrow. Cell nuclei were counterstained with DAPI. Objective 40x, scale bar = 10 μm. (D) immunoblots for PAK2, pPAK2, Nck and NCAD. RASFs pre-treated with Nck siRNA (I) and a nonsilencing siRNA (N) were mechanically strained for 15 and 30 min, the cell surface biotinylated with non-membrane-permeable biotin reagent and purified on streptavidin-conjugated magnetic beads. Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. TL = total lysates from 30 min time point. GAPDH was used to control the purity of the plasma membrane fractions.

Article Snippet: Samples were applied in loading buffer (6x stock solution: 375 mM Tris-HCl, 9% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), 9% β-mercaptoethanol (v/v)), separated by 10% SDS/PAGE, Scientific Reports | (2025) 15:9814 12| https://doi.org/10.1038/s41598-025-94012-2 transferred to nitrocellulose filter, and blocked with 5% milk powder (Roth) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, 0,1% Tween20, pH 7.4) for 30 min at room temperature and incubated with antibodies diluted in 1% milk powder/PBST against phospho-PAK2 (Ser20) (Cell Signaling Technology, #2607, dilution 1:1000), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:1000), Nck1 (Cell Signaling Technology, #2319, 1:1000), PAK2 (R&D Systems, #MAB6849; 1:1000), ADAM15 (R&D Systems, #AF935, 1:1000), OB-Cadherin/Cadherin-11 (Santa Cruz Biotechnology, #sc-365867; 1:1000), N-cadherin (R&D Systems, #AF6426; 1:1000), GAPDH (Santa Cruz Biotechnology, #sc-365062; 1:10.000) overnight at 4 °C and developed using appropriate horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, horse anti-mouse IgG #7076, horse anti-rabbit IgG #7074 (1:2500 each), donkey antiGoat IgG (Thermo Fisher #A15999; 1:2500).

Techniques: Membrane, Staining, Double Staining, Western Blot, Purification, Magnetic Beads, Control, Clinical Proteomics

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 5. Binding of PAK2 to ADAM15/NCAD complex at cell membrane is crucially dependent on ADAM15. (A) IPs of strained RASFs using ADAM15 or NCAD antibodies and detection of PAK2 and Nck. TL = total lysate. IgG, mouse IgG served as background control. (B) cell surface biotinylation and enrichment of membrane fractions on streptavidin beads of strained cells with ADAM15-silenced or nonsilenced (N). Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. (C, D) IPs using ADAM15 antibodies in (C) RASFs with silenced and nonsilenced ADAM15 expression (N) and (D) in chondrocyte cell lines transfected with full length ADAM15 (full-A15) or ADAM15 lacking the cytoplasmic domain (Δcyto). (E, F) analogous to (C, D) IPs using N-cadherin antibodies, showing precipitates of PAK2 and Nck in ADAM15-expressing cells only. (E) right, densitometric evaluation of PAK2 from experiments obtained from 4 donors.

Article Snippet: Samples were applied in loading buffer (6x stock solution: 375 mM Tris-HCl, 9% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), 9% β-mercaptoethanol (v/v)), separated by 10% SDS/PAGE, Scientific Reports | (2025) 15:9814 12| https://doi.org/10.1038/s41598-025-94012-2 transferred to nitrocellulose filter, and blocked with 5% milk powder (Roth) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, 0,1% Tween20, pH 7.4) for 30 min at room temperature and incubated with antibodies diluted in 1% milk powder/PBST against phospho-PAK2 (Ser20) (Cell Signaling Technology, #2607, dilution 1:1000), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:1000), Nck1 (Cell Signaling Technology, #2319, 1:1000), PAK2 (R&D Systems, #MAB6849; 1:1000), ADAM15 (R&D Systems, #AF935, 1:1000), OB-Cadherin/Cadherin-11 (Santa Cruz Biotechnology, #sc-365867; 1:1000), N-cadherin (R&D Systems, #AF6426; 1:1000), GAPDH (Santa Cruz Biotechnology, #sc-365062; 1:10.000) overnight at 4 °C and developed using appropriate horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, horse anti-mouse IgG #7076, horse anti-rabbit IgG #7074 (1:2500 each), donkey antiGoat IgG (Thermo Fisher #A15999; 1:2500).

Techniques: Binding Assay, Membrane, Control, Expressing, Transfection

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 8. CDH11-mediated increased cell invasion can be blocked by CDH11 silencing and transfection with miR-130a-3p mimics. (A, B) RASFs were treated with H19 siRNA or negative nonsilencing siRNA (N) and (A) MTT-based cell viability assays and (B) cells transmigrated through matrigel were stained with DAPI, the whole transwell photographed and all cells counted. (C) upper panel, invaded cells prior transfected with 130a- 3p mimics, CDH11 siRNA or scramble control, shown is the mean ± SD from 5 different RASFs. (C) lower panel, immunoblots for CDH11 from cells transfected and strained as in upper panel. *p < 0.05, **p < 0.005, ***p < 0.0005, Student’s test. (D) diagram of summarized results: mechanical strain results in NCAD/ADAM15- mediated phosphorylation of PAK2, resulting in the downregulation of lncRNA H19 and miR-130a-3p and subsequent upregulation of CDH11, which in turn promotes an aggressive phenotype: increased cell invasion. Inhibition of PAK signaling by PAK inhibitor IPA-3 blocks mechano-induced H19 downregulation and subsequently CDH11 upregulation. In addition, mechano-induced PAK2 phosphorylation is accompanied by recruitment of SH2/SH3 adapter Nck and PAK2 to the NCAD/ADAM15 complex, but binding of Nck/PAK2 is restricted to the cytoplasmic domain of ADAM15. The mechano-induced redistribution of PAK2 to the cell membrane does not occur when Nck is silenced.

Article Snippet: Samples were applied in loading buffer (6x stock solution: 375 mM Tris-HCl, 9% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), 9% β-mercaptoethanol (v/v)), separated by 10% SDS/PAGE, Scientific Reports | (2025) 15:9814 12| https://doi.org/10.1038/s41598-025-94012-2 transferred to nitrocellulose filter, and blocked with 5% milk powder (Roth) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, 0,1% Tween20, pH 7.4) for 30 min at room temperature and incubated with antibodies diluted in 1% milk powder/PBST against phospho-PAK2 (Ser20) (Cell Signaling Technology, #2607, dilution 1:1000), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:1000), Nck1 (Cell Signaling Technology, #2319, 1:1000), PAK2 (R&D Systems, #MAB6849; 1:1000), ADAM15 (R&D Systems, #AF935, 1:1000), OB-Cadherin/Cadherin-11 (Santa Cruz Biotechnology, #sc-365867; 1:1000), N-cadherin (R&D Systems, #AF6426; 1:1000), GAPDH (Santa Cruz Biotechnology, #sc-365062; 1:10.000) overnight at 4 °C and developed using appropriate horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, horse anti-mouse IgG #7076, horse anti-rabbit IgG #7074 (1:2500 each), donkey antiGoat IgG (Thermo Fisher #A15999; 1:2500).

Techniques: Transfection, Staining, Control, Western Blot, Phospho-proteomics, Inhibition, Binding Assay, Membrane