Journal: Cell Death Discovery

Article Title: Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms

doi: 10.1038/s41420-023-01342-z

Figure Lengend Snippet: A MIA PaCa-2 cells were incubated for 24 h with vehicle (Control) or gemcitabine (10 µM) before lysosome labeling using anti-LAMP1 antibody. Nuclei were stained with DAPI. Scale bars , 10 µm. Graph shows numbers of LAMP1 puncta per cell calculated using CellProfiler software. Scatter dot plot shows data as means ± SD; n = 31‒48 cells from three independent experiments. Data were statistically analyzed using unpaired t-tests. B Representative images of live MIA PaCa-2 cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with LysoTracker. C Representative images of live MIA PaCa-2 cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Magic Red. D HeLa cells were incubated for 24 h with vehicle (Control) or gemcitabine (10 µM) before lysosome labeling using anti-LAMP1 antibody. Nuclei were stained with DAPI. Scale bars , 10 µm. Graph shows numbers of LAMP1 puncta per cell calculated using CellProfiler software. Scatter dot plot shows data as means ± SD; n = 22–28 cells from three independent experiments. Data were statistically analyzed using unpaired t -tests. E Representative images of live HeLa cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Lysotracker. F Representative images of live HeLa cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Magic Red. G MIA PaCa-2 cells were incubated with vehicle (−) or gemcitabine (10 µM) for the indicated periods. Total cell lysates were analyzed by immunoblotted using anti-CTSB and β-ACTIN antibodies. Levels of pro-CTSB are barely detectable compared with mature CTSB.

Article Snippet: MIA shNT and MIA shTFEB cells (1 × 10 5 /well) were seeded in duplicate in 12-well plates for 24 h, then incubated with vehicle or 10 μM gemcitabine for 24 h. The activity of CTSB was determined using fluorometric assays (NBP2-54841; Novus Biologicals, LLC, Centennial, CO, USA) as described by the manufacturer.

Techniques: Incubation, Control, Labeling, Staining, Software

Journal: Cell Death Discovery

Article Title: Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms

doi: 10.1038/s41420-023-01342-z

Figure Lengend Snippet: A Quantification of MIA shNT and MIA shTFEB cells incubated for 24 h with vehicle (Control) or gemcitabine (Gem; 10 µM) and stained with LysoTracker. Data are shown as means ± SD of n = 471–2196 cells from three independent experiments and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. B Quantification of MIA shNT and MIA shTFEB cells incubated for 24 h with vehicle (Control) or gemcitabine (Gem; 10 µM) and stained with Magic Red. Data are shown as means ± SD of n = 1220–3156 cells from three independent experiments and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. C Activity of CTSB in MIA shNT and MIA shTFEB cells incubated with vehicle (Control) or gemcitabine (Gem; 10 µM) for 24 h ( N = 6 from three color-coded independent experiments). Data show color-coded independent experiments, means ± SD and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. D MIA shNT and MIA shTFEB cells were incubated for 48 h with vehicle (Control) or gemcitabine (10 µM). Total cell lysates were analyzed by immunoblotting using anti-TFEB, CTSB, and GAPDH antibodies.

Article Snippet: MIA shNT and MIA shTFEB cells (1 × 10 5 /well) were seeded in duplicate in 12-well plates for 24 h, then incubated with vehicle or 10 μM gemcitabine for 24 h. The activity of CTSB was determined using fluorometric assays (NBP2-54841; Novus Biologicals, LLC, Centennial, CO, USA) as described by the manufacturer.

Techniques: Incubation, Control, Staining, Activity Assay, Western Blot

Journal: Toxicological Sciences

Article Title: Generation of a New Model Rat: Nrf2 Knockout Rats Are Sensitive to Aflatoxin B 1 Toxicity

doi: 10.1093/toxsci/kfw065

Figure Lengend Snippet: Antibodies Used in Western Blot

Article Snippet: The antibodies used for Western blot are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 3. caption a7 Antibody Catalog Number Reference or Company Anti-AKR7A2 ab175295 Abcam PLC, Cambridge, United Kingdom Anti-AKR7A3 13209-1-AP Proteintech Group, Inc., Chicago Anti-GSTA3 Mclellan et al. (1994) Anti-Keap1 No. 111 Watai et al. (2007) Anti-Lamin B sc-6217 Santa Cruz Biotechnology Inc., Dallas, Texas Anti-Nrf2 No. 103 Maruyama et al. (2008) Anti-NQO1 ab2346 Abcam PLC, Cambridge, United Kingdom Anti-αTubulin T9026 Sigma-Aldrich Co., LLC, St Louis Open in a separate window Antibodies Used in Western Blot

Techniques: Western Blot

Journal: Viruses

Article Title: Infectious Spleen and Kidney Necrosis Virus Triggers Ferroptosis in CPB Cells to Enhance Virus Replication.

doi: 10.3390/v17050713

Figure Lengend Snippet: Figure 3. ISKNV infection induces ferroptosis in CPB cells. (A) Transmission electron microscopy of CPB cells treated with DMSO (72 h), erastin (10 µmol/L, 72 h), and ISKNV (100 MOI, 24 h, 48 h, 72 h). Scale bars = 1 µm. (B) Analysis of Fe2+ levels in CPB cells after treatment with ISKNV (100 MOI) using the fluorescent probe FerroOrange by laser scanning confocal microscopy. Scale bars = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity of (B) using Image J. (D) Analysis of intracellular ROS levels using DCFH-DA staining, and laser scanning confocal microscopy of CPB cells treated with ISKNV (100 MOI) for 24–72 h. Scale bars = 10 µm. (E) Quantitative analysis of the mean fluorescence intensity of (D) using Image J. (F–H) Detection of Fe2+, ROS, and MDA levels in cell lysates treated with ISKNV (100 MOI) for 24–72 h by microplate reader. * p < 0.05, ** p < 0.01, and *** p < 0.001, with p > 0.05 considered not significant (ns).

Article Snippet: The Fe2+ content of the cells was detected by laser scanning confocal microscopy and a microplate reader using the fluorescent probe FerroOrange (Elabscience, E-BC-F101, Wuhan, China).

Techniques: Infection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Fluorescence, Staining