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: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. (B) The PKN1 TM mutant S916A has reduced kinase activity towards multiple substrates. (C) Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. (D, E) The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. (B) Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. (C) Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. (D) Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. (E) Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (**** p =< 0.0001). (G) Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. (B) PKN1 and PKN2 expression (using microarray data from [47]) in normal prostate, primary tumor, and metastases. (C) RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT and select mTOR components. ** p =< 0.01 *** p =< 0.001 **** p =< 0.0001

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of weaned mice from double heterozygous intercrosses.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: (A) Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β-galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2mm, 0.5mm, 1.0mm. B) Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75 and E9.5. Scale bars 0.2mm (upper four panels), 1.0mm. (C) Whole mount images of wild type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2mm.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Genotypes of embryos from Pkn2 heterozygous intercrosses.

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: The Prostate

Article Title: The Protein Kinase C Super-family Member PKN is Regulated by mTOR and Influences Differentiation During Prostate Cancer Progression

doi: 10.1002/pros.23400

Figure Lengend Snippet: Prostate phenotypes in the ventral lobe

Article Snippet: Plasmids and siRNAs PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with an N-terminal Flag tag.

Techniques:

Journal: Nature Communications

Article Title: Deducing the presence of proteins and proteoforms in quantitative proteomics

doi: 10.1038/s41467-018-04411-5

Figure Lengend Snippet: Select peptide-to-protein clusters with proteins differentially regulated in CFBE41o – vs. HBE41o – cells. a The protein cluster shows the family of related peroxiredoxin (PRDX) proteins. Edges and peptide node outlines in red as well as number associated with the edge in the peptide-to-protein clusters indicate differential expression according to the protein pair-centric analysis. b The protein cluster of serine–threonine kinase-related protein kinases PKN1, PKN2, and PKN3 is displayed. c The peptide-to-protein cluster of Na/K-ATPase proteins is depicted. It is expressed at the same levels in HBE41o – and CFBE41o – cells. d Western blot analysis was used to verify the difference in PKN2 expression levels between HBE41o – and CFBE41o – cells. Na + /K + -ATPase expression levels are shown as a loading control. Data in the western blot represent independent biological replicates, CFBE41o – : n = 2, HBE41o – : n = 2

Article Snippet: PKN2 and NaK-ATPase were detected by incubation with monoclonal anti-PKN2 antibody (dilution 1:1.000, clone 3A7, Novus Biologicals #H00005586-M01) and anti-Na + /K + ATPase antibody H-300 (dilution 1:2000, Santa Cruz #sc28800), respectively, followed by incubation with goat anti-mouse (dilution 1:10,000, Jackson ImmunoResearch Laboratories #205-035-108) or goat anti-rabbit (dilution 1:10,000, Cell Signaling Technology #7074S) IgG antibodies coupled to horse radish peroxidase, respectively.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: A Possible Mechanism behind Autoimmune Disorders Discovered By Genome-Wide Linkage and Association Analysis in Celiac Disease

doi: 10.1371/journal.pone.0070174

Figure Lengend Snippet: Biological functions of genes surrounding the 603 top associated SNPs. Results from IPA.

Article Snippet: PKN2 , Hs00178944_m1 , protein kinase N2 , 1.14 , DOWN , 0.024 , 0.91 , two-locus.

Techniques: Migration

Journal: PLoS ONE

Article Title: A Possible Mechanism behind Autoimmune Disorders Discovered By Genome-Wide Linkage and Association Analysis in Celiac Disease

doi: 10.1371/journal.pone.0070174

Figure Lengend Snippet: The top four networks generated by the Ingenuity IPA software (allowing only direct connections between proteins/genes).

Article Snippet: PKN2 , Hs00178944_m1 , protein kinase N2 , 1.14 , DOWN , 0.024 , 0.91 , two-locus.

Techniques: Generated, Software, Ubiquitin Proteomics, Cell Function Assay, Modification

Journal: PLoS ONE

Article Title: A Possible Mechanism behind Autoimmune Disorders Discovered By Genome-Wide Linkage and Association Analysis in Celiac Disease

doi: 10.1371/journal.pone.0070174

Figure Lengend Snippet: Results from gene expression analysis of 34 candidate genes.

Article Snippet: PKN2 , Hs00178944_m1 , protein kinase N2 , 1.14 , DOWN , 0.024 , 0.91 , two-locus.

Techniques: Gene Expression, Binding Assay, RNA Binding Assay, Transduction, Expressing

Journal: PLoS ONE

Article Title: Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

doi: 10.1371/journal.pone.0013165

Figure Lengend Snippet: Peptides identified by peptide mass fingerprint analysis.

Article Snippet: Clones for in vitro translation have been obtained from ImaGenes GmbH (Berlin, Germany), except PKN1 for which the above mentioned expression clone was used: RSK1 (gi:15929012), RSK2 (gi:109730041), RSK3 (gi:27696716), RSK4 (gi:32450548), PKN2 (gi:30354737), PKN3 (gi:38565959).

Techniques: