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: The Journal of investigative dermatology

Article Title: S-Palmitoylation of Tyrosinase at Cysteine 500 Regulates Melanogenesis.

doi: 10.1016/j.jid.2022.08.040

Figure Lengend Snippet: Figure 5. Intercellular localization of DHHC2, DHHC15, and DHHC3 in NHEMs. NHEMs were transfected with plasmid encoding Myc-tagged DHHC2, DHHC15, or DHHC3. Two days after transfection, they were fixed, permeabilized, and stained with (a) HMB45, (b) calnexin, (c) GM130, or (d) Vti1b antibody and a Myc antibody. Bar ¼ 20 mm. DHHC, Asp-His-His-Cys; NHEM, normal human epidermal melanocyte.

Article Snippet: The antibody against Vti1b (611404; RRID:AB_398926; 1:1,000 for IF) was purchased from BD Transduction Laboratories (Franklin Lakes, NJ; anti-mouse IgG (HþL)-CF 488A secondary antibody [20010; 1:2,000 for IF]) was purchased from Biotium (Fremont, CA), and anti-Rabbit IgG (HþL)-Alexa Fluor 594 secondary antibody (111-586-144; RRID: AB_2338070; 1:2,000 for IF) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Techniques: Transfection, Plasmid Preparation, Staining