Journal: Cancers

Article Title: Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer

doi: 10.3390/cancers12040841

Figure Lengend Snippet: Kinase activity of DDR1 is involved in cohesive cell cluster formation. ( a ) Confocal-microscopic cell images of Ser19-phosphorylated myosin light chain (pMLC (S19)) staining (green) in the indicated OSCC cell lines. Nuclei were counterstained with DAPI (blue). Multi-cell cohesiveness, as evident by decreased pMLC (S19) staining at cell–cell contacts, was only observed in TW2.6 cells. ( b ) Confocal images of pMLC (S19) staining in A-431 (left) and TW2.6 (right) cells treated with vehicle control (VC) or DDR1 inhibitors (10 μM imatinib, 0.1 μM dasatinib, or 10 μM DDR1-IN-1). ( c ) Confocal images of CDH1 staining (red) in A-431 and TW2.6 treated with VC or DDR1 inhibitors. Scale bars, 20 μm.

Article Snippet: The antibodies and reagents used in this study were purchased from the following sources: anti-CDH1 (#3195) and anti-p-MLC (S19) (#3671) was from Cell Signaling (Danvers, MA, USA); anti-α-tubulin (#05-829) and anti-p-Tyr (4G10) (#16-316) was from Millipore (Billerica, MA, USA); anti-DDR1 was from Santa Cruz Biotechnology (sc-532, Santa Cruz, CA, USA); anti-PDPN was from Biocare Medical (CM 266, Pacheco, CA, USA); imatinib mesylate was from Sigma-Aldrich (SML1027, St. Louis, MO, USA); dasatinib was from Selleckchem (S1021, Houston, TX, USA).

Techniques: Activity Assay, Staining, Control

Journal: Mediators of Inflammation

Article Title: AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin

doi: 10.1155/2017/6893560

Figure Lengend Snippet: AM966 induces RhoA activity and phosphorylation of MLC. (a) Confluent HLMVECs were starved 3 h and treated with AM966 (1.0 μ M) and thrombin (1 U/mL) for indicated time periods. Activated RhoA was immunoprecipitated from total lysates by following the manufacturer's instructions (Rho Activation Assay Kit, Millipore). The amount of activated RhoA is determined by a western blot using a RhoA specific antibody. (b) Analysis of activated RhoA by densitometry of the results in (a) were performed by Image J software ( n = 3), and statistical analysis was shown. (c) Confluent HLMVECs were treated with AM966 (1.0 μ M) or LPA (5 μ M) for indicated time periods after 3 h starvation. Cell lysates were immunoblotted with phospho-MLC (P-MLC) and total MLC antibodies. (d) Analysis of P-MLC by densitometry of the results in (c) was performed by Image J software ( n = 3), and statistical analysis was shown. (e) Serum starved confluent HLMVECs were pretreated with Rho kinase inhibitor (10 μ M) for 1 h and then incubated with DMSO or AM966 (1.0 μ M) for an additional 30 min. Lysates were immunoblotted with P-MLC, total MLC, and β -actin antibodies. (f) Analysis of P-MLC by densitometry of the results in (e) was performed by Image J software ( n = 3), and statistical analysis was shown. Shown are representative blots from three independent experiments. (g) Confluent HLMVECs were plated on gold microelectrodes and pretreated with 10.0 μ M Rho kinase inhibitor for 1 h and then stimulated by 1.0 μ M AM966 or DMSO. The TEER tracing represents pooled data (±SEM) from 3 independent experiments. (h) The resistance in response to AM966 treatments during indicated time period (g) was quantified and statistical analysis was performed.

Article Snippet: Phospho (T18/S19)-MLC, MLC, antibodies, and cell lysis buffer were obtained from Cell Signaling.

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Activation Assay, Western Blot, Software, Incubation

Journal: Nature Communications

Article Title: Suppression of endothelial ceramide de novo biosynthesis by Nogo-B contributes to cardiometabolic diseases

doi: 10.1038/s41467-025-56869-9

Figure Lengend Snippet: A Vascular function of mesenteric arteries (MA) was assessed in WT or Nogo-A/B-deficient mice fed with SD or HFD for 6 months by using the pressure myograph system (Danish MyoTechnology, Aarhus, Denmark), (Created in BioRender. Di Lorenzo, A. (2025) https://BioRender.com/i50g226 ). B Ach-mediated vasodilation (WT SD n = 6 mice, Nogo-A/B-deficient SD n = 4 mice, WT HFD n = 6 mice, Nogo-A/B-deficient HFD n = 6 mice); C Flow-induced vasodilation (WT SD n = 6 mice, Nogo-A/B-deficient SD n = 5 mice, WT HFD n = 5 mice, Nogo-A/B-deficient HFD n = 6 mice). HFD MA of WT or Nogo-A/B-deficient mice were incubated with W146 (300 nM, 30 min), an S1P1 inhibitor (WT SD n = 6 mice, Nogo-A/B-deficient SD n = 5 mice, WT HFD n = 5 mice, Nogo-A/B-deficient HFD n = 6 mice, WT HFD n = 4 mice and Nogo-A/B-deficient HFD n = 3 mice treated with W146); D , E PE-induced vasoconstriction (WT SD n = 6 mice, Nogo-A/B-deficient SD n = 4 mice, WT HFD n = 6 mice, Nogo-A/B-deficient HFD n = 6 mice); F myogenic tone (WT SD n = 7 mice, Nogo-A/B-deficient SD n = 4 mice, WT HFD n = 6 mice and Nogo-A/B-deficient HFD n = 6 mice); and G AngII-induced vasoconstriction (WT SD n = 4 mice, Nogo-A/B-deficient SD n = 4 mice, WT HFD n = 4 mice and Nogo-A/B-deficient HFD n = 4 mice) in MA. LC–MS/MS quantification of H total and I specific Cer, and of J total and K specific SM in MA (WT HFD n = 11 mice, Nogo-A/B-deficient HFD n = 9 mice). Data are expressed relative to WT HFD. L WB analysis of p-MLC (Ser19), MLC, p-MYPT1 (Thr696), MYPT1 and NOGO-B in MA from obese WT or Nogo-A/B-deficient mice, with or without AngII stimulation (10 −7 M, 20 s) and M , N relative quantification ( n = 4 mice per group). Data were expressed as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Statistical analysis was performed with two-way ANOVA with Sidak multiple comparisons test ( B – G ) and unpaired t-test two tailed ( H – K , M , N ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: Nogo-B (R&D, #AF6034), Ormdl3 (Millipore, #ABN417), SPTLC1 (BD Biosciences, #611305), SPTLC2 (Abclonal, ##A11716), phospho-S19-MLC, MLC, phospho-T696-MYPT1 (Cell Signaling Technology, #3671, #3672 and 5163 respectively), MYPT1 (sc-514261, SantaCruz Biotechnology) β-Actin (ThermoFisher Scientific, #AM4302).

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice

doi: 10.1172/JCI41749

Figure Lengend Snippet: (A and B) Reexpression of Junb or Myl9 in Junb–/– MEFs restores Myl9 expression, as confirmed by immunoblot analysis of nuclear and total extracts from wild-type MEFs (co) or Junb–/– MEFs that were left untreated or retrovirally transduced. RCC1, Hsc70, and β-actin served as control for equal quality and loading. Results of densitometric quantification of blots are shown. (C and D) Immunofluorescence staining for F-actin and phosphorylated Myl9 (p-Myl9) on Junb–/– MEFs transduced with an empty vector or Junb- or Myl9-encoding retroviral expression vector. Scale bars: 10 μm. In C and D, one representative staining of at least 3 independent cell preparations is shown (n ≥ 3). (E and F) Relative wound closure of control and Junb–/– MEFs and of the same cells transduced with empty vector or Junb- or Myl9-containing vector subjected to scratch wounding. Values were calculated from images of the wound area taken at 0, 8, and 18 hours after wounding. ***P < 0.005. (G) Relative collagen gel contraction by wild-type or Junb–/– MEFs transduced with empty vector or Junb- or Myl9-encoding retroviral expression vector. Gel contraction capability was determined 96 hours after seeding as percentage of the initial gel surface area that was set to 100%. Results represent the mean ± SD of 3 independent experiments (n = 3), each performed in triplicate (E and G). Bonferroni multiple comparisons test was applied for statistical analysis. ***P < 0.001.

Article Snippet: Antibodies used were anti-Junb (1:500, N17, Santa Cruz Biotechnology Inc.), anti-RCC1 (1:500; BD Biosciences), anti-Hsc70 (1:10,000, Stressgene), anti–β-actin (1:1,000, Santa Cruz Biotechnology Inc.), anti–phospho–MLC-2-S19 (1:1,000, Cell Signaling Technology), anti–MLC-2 (1:1,000, Cell Signaling Technology), anti-FAK (1:1,000, Cell Signaling Technology), anti–phospho–FAK-Y397 (1:1,000, Biosource), and anti–phospho–FAK-Y576/577 (1:1,000, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Transduction, Plasmid Preparation