Journal: STAR Protocols

Article Title: A protocol to characterize zebrafish LGP2 as a dual regulator of IFN response during viral infection

doi: 10.1016/j.xpro.2022.101844

Figure Lengend Snippet:

Article Snippet: medium 199 (Gibco) , HyClone , Cat# SH30253.01.

Techniques: Recombinant, Protease Inhibitor, Staining, Lysis, Western Blot, Plasmid Preparation, Isolation, Purification, SYBR Green Assay, Reporter Assay, Expressing, Software, Dissection

Journal: Antioxidants

Article Title: Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux

doi: 10.3390/antiox11051009

Figure Lengend Snippet: Diclofenac induces microtubule depolymerization. ( A ) Confocal microscopy of HeLa cells stained with antibodies to α-tubulin (green) after incubation at 37 °C (upper panel) for 6 h or at 37 °C for 3 h followed by 4 °C for 3 h. Cells were incubated in a medium containing vehicle (0.1% dimethyl sulfoxide [DMSO]), nocodazole (100 nM), taxol (100 nM), or diclofenac (170 µM, 200 µM). Nuclei were stained with 4–6-diamidino-2-phenylindole (DAPI, blue). ( B ) Quantitative analysis of mean fluorescence intensity of α-tubulin. Data are presented as means ± SD from three independent experiments ( n = 52–60 cells). * p < 0.05; ** p < 0.01 (Student’s t -test). ( C ) Confocal microscopy of HepG2 cells stained with antibodies to α-tubulin (green) and LAMP1 (red) after incubation in a medium containing vehicle (0.1% DMSO) or diclofenac (500 µM) under fed (Dulbecco’s modified eagle medium, 10% fetal bovine serum) conditions or starved (Earle’s balanced salt solution) conditions for 8 h. Nuclei were stained with DAPI (blue). Representative images are shown. ( n = 14–18 cell). Areas enclosed by the white boxes are shown at higher magnification. Yellow boxes indicate the edges of the plasma membrane. Three independent experiments were performed. Scale bar, 10 µm; scale bar in magnification; 2 µm. ( D ) In vitro tubulin polymerization. Polymerization activity was monitored in the presence of DMSO (0.01%, vehicle), taxol (10 μM), nocodazole (10 μM), or diclofenac (0.17 mM and 1.7 mM) for 30 min at 37 °C as the increase in A 340 nm. Two independent experiments were performed.

Article Snippet: For starvation to induce autophagy, cells were cultured in Earle’s balanced salt solution (EBSS; CA006-050, GenDEPOT, Katy, TX, USA).

Techniques: Confocal Microscopy, Staining, Incubation, Fluorescence, Modification, Clinical Proteomics, Membrane, In Vitro, Activity Assay

Journal: Antioxidants

Article Title: Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux

doi: 10.3390/antiox11051009

Figure Lengend Snippet: Diclofenac inhibits the fusion of autophagosomes and lysosomes. ( A ) Confocal microscopy of HepG2 cells expressing mCherry-GFP-LC3 were incubated in a medium containing vehicle (0.1% dimethyl sulfoxide [DMSO]), Earle’s balanced salt solution (EBSS), rapamycin (0.25 µM), diclofenac (500 µM), or bafilomycin A1 (100 nM) for 8 h. Autophagosomes appear as yellow spots (mCherry + /GFP + ) and autolysosomes appear as red spots (mCherry + /GFP − ) in merged images. Nuclei were stained with 4–6-diamidino-2-phenylindole (DAPI, blue). ( B ) Quantification of autophagic vesicle number as the sum of autophagosome (mCherry + /GFP + , yellow) spot and autolysosome (mCherry + /GFP − , red) spot number per cell. ( C ) Ratio of number of autophagosomes (AP) to autolysosomes (AL) per cell. ( D ) Size of autophagosomes and autolysosomes was quantified by diameter, perimeter, length, and width of each spot. Data are presented as means ± SD from three independent experiments ( n = 15 cells). ( E ) HepG2 cells were incubated in a medium containing vehicle (0.1% DMSO), EBSS, rapamycin (0.25 µM), diclofenac (500 µM), or bafilomycin A1 (100 nM) for 8 h. Total cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Uncropped western blots in ( F ) The relative immunoblot intensities of LC3-II, p62, and mTor normalized by those of actin were also determined as means ± SD from three independent experiments. ( G ) Nutrient-starved (EBSS-treated) HepG2 cells expressing GFP-LC3 were incubated in a medium containing vehicle (0.1% DMSO), diclofenac (500 µM), or bafilomycin A1 (100 nM) for 8 h. Cells were then subjected to immunofluorescence analysis with antibodies to LAMP1 (lysosomal marker, red) and observed using confocal microscopy. Nuclei were stained with DAPI. ( H ) Quantification of the ratio of LC3 spots in the lysosome (trapped LC3 spots) to total LC3 spots. Lower values on the Y-axis indicate that a small number of LC3 spots exist in the lysosomes. Data are presented as means ± SD from three independent experiments ( n = 29–39 cells). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -test). Scale bar, 20 µm; scale bar in inset, 2 µm.

Article Snippet: For starvation to induce autophagy, cells were cultured in Earle’s balanced salt solution (EBSS; CA006-050, GenDEPOT, Katy, TX, USA).

Techniques: Confocal Microscopy, Expressing, Incubation, Staining, Western Blot, Immunofluorescence, Marker

Journal: Antioxidants

Article Title: Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux

doi: 10.3390/antiox11051009

Figure Lengend Snippet: Diclofenac inhibits phagophore movement followed by autophagosome formation. ( A ) Nutrient-starved (Earle’s balanced salt solution) HepG2 cells expressing GFP-LC3 and mCherry-WDFY (PtdIns(3)P reporter) were incubated in a medium containing vehicle (0.1% dimethyl sulfoxide), diclofenac (500 µM), or SAR405 (500 nM) for 8 h. Nuclei were stained with 4–6-diamidino-2-phenylindole. Images were obtained from confocal microscopy. Scale bars, 20 µm; scale bar in insets, 2 µm. ( B ) Quantification of images from ( A ). The number of LC3 spots (left) and WDFY spots (middle) per cell. Relative sum intensity of WDFY fluorescence per cell (right) was measured, which reveals all PtdIns(3)P signals including aggregates as shown in diclofenac-treated cells. Data are presented as means ± SD from three independent experiments ( n = 20–28 cells). *** p < 0.001 (Student’s t -test).

Article Snippet: For starvation to induce autophagy, cells were cultured in Earle’s balanced salt solution (EBSS; CA006-050, GenDEPOT, Katy, TX, USA).

Techniques: Expressing, Incubation, Staining, Confocal Microscopy, Fluorescence

Journal: Antioxidants

Article Title: Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux

doi: 10.3390/antiox11051009

Figure Lengend Snippet: Diclofenac induces fragmentation of mitochondria and the Golgi during cell death. ( A , C ) Fed ( A, Dulbecco’s modified eagle medium, 10% fetal bovine serum) or nutrient-starved ( C , Earle’s balanced salt solution) HepG2 cells were incubated in a medium containing vehicle (0.1% dimethyl sulfoxide [DMSO]), n -acetylcysteine (2 mM), diclofenac (500 µM), or diclofenac (500 µM) with n -acetylcysteine (2 mM) for 8 h. Cells were stained with Mitotracker dye to measure mitochondria morphology. Images were obtained from confocal microscopy. Red line represents mitochondrial morphological skeleton for quantification. Scale bars are shown in each image. ( B , D ) Quantitative analysis for mitochondrial fragmentation. Ratio of mean rod length to branch length or that of median rod length to branch length of mitochondrial skeleton network per cell was calculated using the Mitochondrial Network Analysis toolset applied with Image J. Data are presented as means ± SD from three independent experiments ( n = 9–13 cells). ( E , F ) Detection of mitochondrial reactive oxygen species using MitoSOX red. HepG2 cells were incubated in a medium containing vehicle (0.1% DMSO), rotenone (100 µM), rapamycin (0.25 µM), or diclofenac (500 µM) for 8 h. Cells were then imaged using a high-content imaging system (ImageXpress Confocal HT.ai) and red fluorescence mean intensity per cell was quantified. Data are presented as means ± SD from three independent experiments ( n = 1589–2574 cells). Scale bar, 50 µm. ( G ) HepG2 cells were incubated in a medium containing vehicle (0.1% DMSO), rotenone (100 µM), rapamycin (0.25 µM), or diclofenac (500 µM) for 8 h. Cells were then subjected to immunofluorescence analysis with antibodies to Golgin97 (red). Nuclei were stained with 4–6-diamidino-2-phenylindole. Images were obtained from confocal microscopy. Scale bar, 20 µm; scale bar in inset, 2 µm. ( H ) Relative mean intensity of Golgin97 fluorescence in the perinuclear region of the cell is presented as means ± SD from three independent experiments (upper histogram, n = 42–50 cells). Cell viability assay using a water-soluble tetrazolium salt (EZ-cytox) is presented as means ± SD from three independent experiments (lower histogram). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -test).

Article Snippet: For starvation to induce autophagy, cells were cultured in Earle’s balanced salt solution (EBSS; CA006-050, GenDEPOT, Katy, TX, USA).

Techniques: Modification, Incubation, Staining, Confocal Microscopy, Imaging, Fluorescence, Immunofluorescence, Viability Assay