Journal: Biochemical Journal

Article Title: Opposite effects of spermidine and GC7 in cell culture are dictated by distinct molecular targets

doi: 10.1042/BCJ20253298

Figure Lengend Snippet: ( A ) Representative confocal images of HCT116 cells transduced with the lentivirus expressing the mCherry-GFP-LC3, pre-treated with 1 mM DFMO for 72 hours, then treated with the indicated concentrations of SPD for 6 hours (top) or 24 hours (bottom). Yellow puncta indicate autophagosomes (mCherry+/GFP+), while autolysosomes appear red (mCherry+/GFP−). Scale bar 40 μm. ( B ) Western blot analysis showing hypusinated eIF5A (Hyp-eIF5A), total eIF5A, and Vinculin (loading control) in HCT116 cells pre-treated with 1 mM DFMO and the indicated concentrations of SPD for 6 hours (left) or 24 hours (right). ( C ) Representative confocal images of LoVo cells transduced with the lentivirus expressing the mCherry-GFP-LC3, pre-treated with 1 mM DFMO for 72 hours, then treated with the indicated concentrations of SPD for 6 hours (top) or 24 hours (bottom). Yellow puncta indicate autophagosomes (mCherry+/GFP+), while autolysosomes appear red (mCherry+/GFP−). Scale bar 40 μm. ( D ) Western blot analysis showing hypusinated eIF5A (Hyp-eIF5A), total eIF5A, and Vinculin (loading control) in LoVo cells pre-treated with 1 mM DFMO and the indicated concentrations of SPD for 6 hours (left) or 24 hours (right). ( E ) Left: representative confocal images of HCT116 cells treated for 6 hours with 1, 20, or 1000 μM SPD after 72 hours of pre-treatment with 1 mM DFMO. Before imaging, live cells were stained with Lysotracker Red (red puncta) and DAPI (blue). Scale bar 40 μm. Right: quantification of Lysotracker Red signal using ImageJ from 6 independent images normalized to the respective cell number. For statistical analysis, ** P <0. was determined by Dunnett’s multiple comparisons test following one-way ANOVA. ( F ) Right: representative confocal images of HCT116 cells treated for 24 hours with 1, 20, or 1000 μM SPD after 72 hours of pre-treatment with 1 mM DFMO. Before imaging, live cells were stained with Lysotracker Red (red puncta) and DAPI (blue). Scale bar 40 μm. Right: quantification of Lysotracker Red signal using ImageJ from six independent images normalized to the respective cell number. For statistical analysis, ns = not significant ( P >0.05) was determined by Dunnett’s multiple comparisons test following one-way ANOVA.

Article Snippet: Lysotracker Red was purchased by Medchem Express (#HY-D1300) and used according to the manufacturer’s specifications.

Techniques: Transduction, Expressing, Western Blot, Control, Imaging, Staining

Journal: Toxicology and applied pharmacology

Article Title: Phagolysosome Acidification is Required for Silica and Engineered Nanoparticle-induced Lysosome Membrane Permeabilization and Resultant NLRP3 Inflammasome Activity

doi: 10.1016/j.taap.2017.01.012

Figure Lengend Snippet: (A) Cathepsin activity in the whole lavage fluid of C57Bl/6 following instillation of PBS or silica (40 mg/kg) (N=4). (B) Representative LSC images (40X scan) and graph showing Mean Fluorescent Intensity (MFI per total cells counted) of cathepsin B in lung lavage cells isolated from PBS and silica exposed mice (N=4, 2 mice pooled for each N). (C) Histogram overlays of cathepsin activity substrate integrals from combined experiments. (D) Representative LSC images (60X scan) of Lysotracker Red™ staining with figure showing MFI of Lysotracker Red™ for combined experiments (N=4, 2 mice pooled for each N). (D) Histogram overlays of Lysotracker Red™ MFI integrals from combined experiments. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significance.

Article Snippet: Immunofluorescences Detection of Cathepsin B and Lysotracker™ Red Cathepsin B and lysosomes were measured using a CompuCyte iCys Laser Scanning Cytometer (LSC, Westwood, MA, USA).

Techniques: Activity Assay, Isolation, Staining

Journal: Journal of cardiovascular pharmacology

Article Title: Activation of Gα q in cardiomyocytes increases Vps34 activity and stimulates autophagy

doi: 10.1097/FJC.0000000000000461

Figure Lengend Snippet: Mice were injected with tamoxifen for 7 days. (A) Isolated cardiomyocytes loaded with LysoTracker Red were imaged by confocal microscopy (63x magnification). Representative cells are shown. (B) LysoTracker-positive puncta were analyzed using CellProfiler 2.0. Left panel, lysosomes per cell; n=41 for WT and n=64 for QL. Right panel, median lysosome area per cell; n=43 for WT and n=64 for QL. Bars, mean values. p<0.0001, Mann-Whitney test (both panels). (C) Lysosomal proteins in ventricular lysates were detected by western blotting. GAPDH is a loading control.

Article Snippet: LysoTracker Red staining and confocal microscopy Cardiomyocytes isolated as described above were plated on laminin-coated glass-bottom dishes (MatTek Corporation).

Techniques: Injection, Isolation, Confocal Microscopy, MANN-WHITNEY, Western Blot, Control