Journal: Virology

Article Title: Non-productive HIV-1 infection of human glomerular and urinary podocytes.

doi: 10.1016/j.virol.2010.09.005

Figure Lengend Snippet: Fig. 1. HIV-1 enters podocytes by a dynamin-dependent endocytosis. (A) Differentiated AB 8/13 podocytes were incubated for 90 min with fluorescently labeled HIV-1 AD8, washed and incubated for 30 min with LysoTracker Red (LT, 50 nM) or Transferrin-Alexa Fluor 594 (TF, 5 μg/ml). Separate and merged confocal microscopy images are shown. (B) Podocytes were pretreated for 1 h with 200 μM dynasore in 0.1% DMSO (+Dynasore) or with 0.1% DMSO alone (−Dynasore) and infected with AD8 as described in (A). Confocal images of AD8 entry and Transferrin-Alexa Fluor 594 uptake in the absence or presence of dynasore are shown. (C, D) Colocalization of HIV-1 particles (multiplicity of infection 100-fold lower than in Figs. 1A and B) with LysoTracker Red (LT) and Transferrin-Alexa Fluor 594 (TF) in podocytes (C) and TZM-bl cells susceptible to HIV-1 infection (D).

Article Snippet: When indicated, the cells were pretreated for 1 h with 200 μM dynasore (Tocris Bioscience) (Macia et al., 2006) in 0.1% DMSO or with 0.1% DMSO alone and subsequently infectedwith HIV-1 in the presence or absence of dynasore.

Techniques: Incubation, Labeling, Confocal Microscopy, Infection

Journal:

Article Title: Astn2 , A Novel Member of the Astrotactin Gene Family, Regulates the Trafficking of ASTN1 During Glial-Guided Neuronal Migration

doi: 10.1523/JNEUROSCI.0032-10.2010

Figure Lengend Snippet: (A) A granule cell culture migration assay demonstrates that the effects of a soluble noncompetitive inhibitor of Dynamin, Dynasore on neuronal migration are reversible. Dissociated cerebellar granule neurons, labeled with a Venus encoding retrovirus, were imaged for three 60 minute periods; vehicle (DMSO) or DMSO and either 40 µM or 80 µM Dynasore were added during the second imaging period. To remove the Dynasore, we washed the cells three times with granule cell medium, and imaged migrating neurons as above for 1 hr. The images in (A) are inverted for better resolution and show migrating granule neurons labeled with Venus. In this study, we selected neurons with the features of actively migrating neurons, i.e. an elongated cell somata that was apposed to and flattened against Bergmann-like glial fibers. Migrating neurons are labeled by number in the 0 hr. panel and their final position is indicated by the same number in the 1 hr. panel. The number of cells that migrated more than 5 µm/hr were counted and expressed as a percentage of total labeled cells. (B) Cerebellar slices transfected with Venus expressing retrovirus were treated with Dynasore at 40 µM or 80 µM concentration, or with control vehicle (DMSO). TuJ1 staining reveals the parallel fiber layer of differentiated granule neurons in the inner EGL. In slices incubated with Dynasore, Venus expressing neurons seem to arrest in the EGL compared to DMSO control cells. DRAQ5 was used to counterstain nuclei. Quantitation of neurons in organotypic cerebellar slices shows that neurons remain significantly closer to the pial surface in the presence of 40 or 80 µM Dynasore, relative to controls (*p<0.001). (C) In slices double immunostained with antibodies to GFP and Caspase 3, 48 hr. incubation with Dynasore did not significantly alter the percentage of GFP positive cells that also were Caspase 3 positive. Significance was assessed via Students-t-test; values are mean ± SEM (*p<0.001). Scale bar in (A) and (B) is 50 µm.

Article Snippet: To assay the effect of Dynasore on GCP migration, the organotypic slice cultures were processed for immunohistochemistry with antibodies against EGFP and TUJ1, as described ( Solecki et al., 2001 ) and with DRAQ5 (Cell Signaling Technology, Inc., Danvers, MA) to visualize nuclei.

Techniques: Cell Culture, Migration, Labeling, Imaging, Transfection, Expressing, Concentration Assay, Control, Staining, Incubation, Quantitation Assay

Journal: Interdisciplinary information sciences

Article Title: CRISPR/Cas9 Screenings Reveal the Role of STX1A and CDK1 in Cathepsin G Entering and Killing Colorectal Cancer Cells

doi: 10.4036/iis.2025.A.11

Figure Lengend Snippet: CDK1 inhibitor attenuates the cell-killing function of CTSG. (A) Cas9-expressing DLD1 cells were infected with genome-wide pooled gRNA lentivirus. Infected cells were selected with puromycin for 3 days. The cells were then reseeded and treated with NETs condition medium or DLD1 condition medium for 16 h. Genomic DNA was extracted from DLD1 cells after treatment, and gRNA sequences were amplified by PCR. PCR products were purified and subjected to next-generation sequencing. The differentially expressed genes were plotted based on rank and Beta score. (B) HCT116 and DLD1 cells were treated with 5 ng/μL of CTSG for 24 h with Ro-3306 at the indicated concentrations. The data presented are representative images of cellular morphology. (C) HCT116 cells were treated with 5 ng/μL of CTSG for 16 h with Ro-3306 at the indicated concentrations (ng/μL). The data presented are Western blots of cleaved PARP.

Article Snippet: Dynasore (#S8047) and Ro-3306 (#S7747) were purchased from Selleckchem.

Techniques: Expressing, Infection, Genome Wide, Amplification, Purification, Next-Generation Sequencing, Western Blot

Journal: Cells

Article Title: Transfer of Cardiac Mitochondria Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in a Preclinical Model of Ischemic Heart Disease.

doi: 10.3390/cells12040582

Figure Lengend Snippet: Figure 2. Cardiac mitochondria are internalized by MSCs through dynamin-dependent, clathrin- mediated endocytosis. (A) Representative confocal microscopy pictures of WGA-stained MSCs after 24 h of incubation with MitoTracker Green-labeled cardiac mitochondria at the Mito 3 concentration in the absence or presence of dynasore. Scale bar: 5 µm. (B) Flow cytometry quantification of Mito- Tracker Green-labeled cardiac mitochondria by MSCs following 24 h of exposure in the presence or absence of dynasore (n = 4). (C) Relative Ki67 mRNA levels in cardiac mitochondria-preconditioned MSCs in the presence or absence of dynasore in reference to their respective controls (n = 4). (D) Relative VEGF and HGF mRNA levels in cardiac mitochondria-preconditioned MSCs in the presence or absence of dynasore in reference to their respective controls (n = 4). (E) Relative mRNA levels of CXCL1, CXCL5, CXCL6, IL11, IL33 and LIF in cardiac mitochondria-preconditioned MSCs in the presence or absence of dynasore in reference to their respective controls (n = 4). (F) Relative mRNA levels of MMP1, MMP9 and MMP14 (n =4) and (G) collagenase activity (n = 10) in cardiac mitochondria-preconditioned MSCs in the presence or absence of dynasore in reference to their respective controls. One-way ANOVA with Dunn’s multiple comparisons test in (B–F). One-way ANOVA with Tukey’s multiple comparisons test in (G). * p < 0.05, ** p < 0.01, *** p < 0.001. Each dot represents an independent experiment. Bar graphs represent mean values ± SD.

Article Snippet: To characterize the endocytosis process by which MSCs internalize cardiac mitochondria, human MSCs were exposed to cardiac mitochondria previously labeled with MitoTracker Green FM (40 nM, Invitrogen, Waltham, MA, USA, Cat#M7514) in the presence of the dynamin-dependent, clathrin-mediated endocytosis inhibitor dynasore (50mM, Santa Cruz Biotechnology, Dallas, TX, USA, Cat#sc-202592).

Techniques: Confocal Microscopy, Staining, Incubation, Labeling, Concentration Assay, Flow Cytometry, Activity Assay