Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet: Morphological changes in the cytoskeleton of dysfunctional VSMCs Scale bar: 20 μm

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques:

Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet: Schematic workflow supernatant assay Immunofluorescence staining shows microglia activation after incubation with supernatant from dysfunctional VSMCs. Activated microglial were characterized by morphologic changes and elevated expression of IL-6 at 100 × magnification, scale bars: 5 μm

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques: Immunofluorescence, Staining, Activation Assay, Incubation, Expressing

Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet:

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques: Recombinant, Wound Healing Assay, Staining, SYBR Green Assay, Software, Real-time Polymerase Chain Reaction, Microscopy, Sterility, Inverted Microscopy, Confocal Microscopy

Journal: iScience

Article Title: Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

doi: 10.1016/j.isci.2024.109779

Figure Lengend Snippet:

Article Snippet: CD163 human antibody (clone GHI/61.1) , Miltenyi , Cat#130-123-249; RRID: AB_2819455.

Techniques: Recombinant, Adhesive, Sequencing, Modification, DC Protein Assay, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Gene Expression, Software, Saline

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A – D ) ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with Midostaurin (100 nM) for additional 18 hr. Cell migration and invasion were tested by transwell ( A, B ) or wound healing ( C, D ) assay. n=5 biologically independent replicates. Scale bar = 500 µm ( A ) and 100 µm ( C ). Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. * p<0.05, ** p<0.01, and *** p<0.001 . Figure 5—figure supplement 6—source data 1. PKC inhibition counteracts PLCE1-induced cell migration and invasion.

Article Snippet: An intracellular calcium chelator BAPTA-AM (HY-100545), a PKC inhibitor Midostaurin (HY-10230), and an IP3R antagonist (HY-W009724) were obtained from MedChemExpress (NJ, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Migration, Two Tailed Test, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) IP3R blocking inhibits PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with 2-APB (10 µM) for additional 18 hr. ( B ) [Ca 2+ ] blocking represses signal transduction in the axis of PKC/GSK-3β/β-Catenin induced by PLCE1. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with BAPTA-AM (10 µM) for additional 18 hr. ( C ) PKC inhibition blocks PLCE1 stimulated signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 . 6 hr post-transfection, cells were treated with 100 nM of Midostaurin for additional 18 hr. ( D ) PKC inhibition represses PLCE1-induced nuclear translocation of β-Catenin in ECA-109 cells. Cells were transfected with the plasmid expressing PLCE1. 6 hr post-transfection, 2-APB (10 µM), BAPTA-AM (10 µM), or Midostaurin (100 nM) was added in culture medium, and cells were cultured for additional 18 hr. Scale bar = 10 µm. Immunofluorescence was used to examine subcellular distribution of β-Catenin. ( E ) TAK1 counteracts PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated for 24 hr. ( F ) TAK1 reduces PLCE1-induced nuclear distribution of β-Catenin in ECA-109 cells. Cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated. Scale bar = 10 µm. ( G ) Dominant negative TAK1 (K63W) fails to block signal transduction in the axis of PKC/GSK-3β/β-Catenin/MMP2 induced by PLCE1. ECA-109 cells were transfected with the plasmids expressing Plce1 or mutated Map3k7 (TAK1 K63W) for 24 hr. ( H ) TAK1 has no effect on PLCE1 S1060A-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing PLCE1 S1060A or TAK1 for 24 hr. Protein levels were analyzed by western blot, and Actin was used as a loading control. Representative blots were shown. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot analysis displayed in .

Article Snippet: An intracellular calcium chelator BAPTA-AM (HY-100545), a PKC inhibitor Midostaurin (HY-10230), and an IP3R antagonist (HY-W009724) were obtained from MedChemExpress (NJ, USA).

Techniques: Blocking Assay, Transduction, Transfection, Plasmid Preparation, Expressing, Inhibition, Translocation Assay, Cell Culture, Immunofluorescence, Dominant Negative Mutation, Western Blot, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet:

Article Snippet: An intracellular calcium chelator BAPTA-AM (HY-100545), a PKC inhibitor Midostaurin (HY-10230), and an IP3R antagonist (HY-W009724) were obtained from MedChemExpress (NJ, USA).

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Software