Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

Journal: Journal of Cell Science

Article Title: The DISC1–Girdin complex – a missing link in signaling to the T cell cytoskeleton

doi: 10.1242/jcs.242875

Figure Lengend Snippet: Girdin in Jurkat cells. (A) DISC1 immunoprecipitates (IP) were probed on blots for Girdin and DISC1. Beads lacking DISC1 antibody (Neg) showed no obvious nonspecific binding. Analysis of the supernatant (Sup) from the IP showed that some Girdin still remained but DISC1 was not detected. Both DISC1 and Girdin were present in the supernatant of lysates treated with beads only. (B–D) WT Jurkat cells (B), Girdin-KO cells (C) and Girdin-KO cells transfected with an Girdin-eGFP construct (D) were paired with SEE-coated Raji cells and fixed. Each cell type was then stained for actin with phalloidin-TRITC and immunostained for Girdin using an FITC secondary Ig. Experiment A was performed twice and experiments B–D were performed three times. (E) The fluorescence intensity of phalloidin-TRITC staining was plotted as the mean±s.e.m. (n=30). A one-tailed Student's t-test showed that the signal intensity of the first five bands was significantly different between WT and Girdin-KO cells (P<0.001) but not between WT and Girdin-KO cells expressing Girdin-eGFP (P>0.05). R, Raji cell. Scale bars: 10 μm.

Article Snippet: The Cas9, DISC1 and Girdin sgRNA plasmids were obtained from Genecopoeia (Cat # CP-LvC9NU-02-B, HCP268459-LvSG03-1-B and HCP259879-LvSG03-1-B).

Techniques: Binding Assay, Transfection, Construct, Staining, Fluorescence, One-tailed Test, Expressing

Journal: Journal of Cell Science

Article Title: The DISC1–Girdin complex – a missing link in signaling to the T cell cytoskeleton

doi: 10.1242/jcs.242875

Figure Lengend Snippet: Jurkat cells on anti-TCR-coated coverslips. (A–J) To monitor the movements of NDE1, LIS1, and dynein during Jurkat cell activation, cells were settled on coverslips coated with anti-TCR Ig. After 15 min incubation, cells were fixed and then some cells were immunostained for DIC using a FITC secondary Ig (A–E) whereas others were immunostained for NDE1 using an AlexaFluor 594 secondary Ig and LIS1 using an FITC secondary Ig (F–J). Each set of immunostaining procedures for either DIC or NDE1 and LIS1 was repeated for WT Jurkat cells (A,F), DISC1-KO cells (B,G), DISC1-KO cells expressing isoform L-eGFP (C,H), DISC1-KO cells expressing DISC1Lv-eGFP (D,I) and Girdin-KO cells (E,J). Merged composite images show NDE1 in red and LIS1 in green. (K) Fluorescence images were classified as having a ring-like staining pattern (Ring), a central patch (Spot) or neither pattern (Misc) from the interface in NDE1-stained cells (n=30). Results were averaged from multiple experiments and plotted as mean±s.e.m. Chi-squared tests were used to show the significant difference in ring formation between WT and DISC1-KO cells (P<0.001), WT and DISC1Lv-eGFP-expressing DISC1-KO cells (P<0.001), or WT and Girdin-KO cells (P<0.001). WT and L-eGFP-expressing DISC1-KO cells were not found to be significantly different (P>0.05). (L,M) WT Jurkat (L) and DISC1-KO (M) cells were settled onto Vβ8-coated coverslips, immunostained for NDE1 and talin, and imaged using confocal microscopy. Experiments A–J were performed four times and experiment L,M was performed twice. Scale bars: 10 μm.

Article Snippet: The Cas9, DISC1 and Girdin sgRNA plasmids were obtained from Genecopoeia (Cat # CP-LvC9NU-02-B, HCP268459-LvSG03-1-B and HCP259879-LvSG03-1-B).

Techniques: Activation Assay, Incubation, Immunostaining, Expressing, Fluorescence, Staining, Confocal Microscopy