Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: BITC increased the expression levels of LC3B and p62 proteins AGS cells. (A) AGS cells were treated with 10 µM BITC for 8, 12, or 24 h. Whole cell lysate was prepared, and LC3B and p62 levels were determined by western blotting. β-actin was used as a loading control. (B) AGS cells were treated with 5, 10, or 15 µM BITC for 24 h. Whole cell lysate was prepared, and LC3B and p62 levels were determined by western blotting. β-actin was used as a loading control. (C) AGS cells were treated with 5, 10, or 15 μM BITC for 12 h and mRNA level was assessed by q-PCR. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01, and *** p <0.001 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Control

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: BITC-induced LC3B conjugation in AGS cells was not dependent on class III PI3K. (A) For the single treatment, AGS cells were treated with 2.5, 5, 10, or 15 μM BITC for 24 h. For the combined treatment, the cells were pre-treated with 0.5 µM wortmannin for 2 h followed by 10 µM BITC for 24 h. Then, the whole cell lysate was prepared, and the class III PI3K protein level was measured by western blotting. β-actin was used as a loading control. (B) The cytotoxicity of wortmannin was detected by MTT assay after treating the cells with different concentrations of wortmannin for 24 and 48 h. (C) AGS cells were pre-treated with 0.25 or 0.5 µM wortmannin for 2 h followed by 10 or 15 µM BITC for 24 h. After cell lysis, the change in LC3B II level was detected by western blotting. β-actin was used as a loading control. Data were calculated from three independent experiments and expressed as the mean ± SEM. ** p <0.01 and *** p <0.001 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Conjugation Assay, Western Blot, Control, MTT Assay, Lysis

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: Additive effect of BITC and bafilomycin A1 on LC3B accumulation in AGS cells. (A) AGS cells were treated with BITC with or without 100 nM bafilomycin A1 for 24 h. Whole cell lysate was prepared, and the LC3B II level was measured by western blotting. β-actin was used as a loading control. (B) Increased LC3B protein expression was further confirmed by immunostaining with anti-LC3B antibody and was detected via Alexa Flour 488-conjugated secondary antibody (green) under a fluorescence microscope (scale-bar=50 μm). DAPI (blue) was used for nuclear staining. (C) The cytotoxicity of bafilomycin A1 was investigated by MTT assay after treating the cells with different concentrations of Bafilomycin A1 for 24 and 48 h. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001 versus the control, and # p <0.05 versus BITC treatment alone.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Western Blot, Control, Expressing, Immunostaining, Fluorescence, Microscopy, Staining, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: No relationship between BITC-induced LC3 B accumulation and cytotoxicity. (A) AGS cells were transfected with 50 nM LC3B siRNA or control siRNA for 12 h and incubated with 10 µM BITC for 8 h. Whole cell lysate was prepared, and siRNA efficacy was confirmed by detecting the LC3B protein expression via western blotting. β-actin was used as a loading control. (B) AGS cells were transfected with 50 nM LC3B siRNA or control siRNA for 12 h, followed by 10 µM BITC treatment for 24 h. Cell viability changes were evaluated by MTT assay. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Transfection, Control, Incubation, Expressing, Western Blot, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: The proposed mechanism of BITC-induced cytotoxicity through autophagy inhibition and lysosomal dysfunction in AGS cells. In human gastric adenocarcinoma AGS cells, BITC induced cell death by caspase-dependent apoptosis and inhibition of cytoprotective autophagy. It inhibited autophagy degradation through lysosomal dysfunction, which decreased both the quality and quantity of cathepsin proteins. Moreover, BITC may inhibit the autophagy initiation and elongation steps, since it decreased the expression levels of Beclin1, Atg5-Atg12 complex, and class III PI3K proteins. However, because of increased mRNA transcription and decreased lysosomal degradation, BITC also caused the accumulation of LC3B and p62. Nevertheless, there was no direct relationship between BITC-induced LC3B II accumulation and cytotoxicity.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing

Journal: PLoS ONE

Article Title: Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line

doi: 10.1371/journal.pone.0165224

Figure Lengend Snippet: (A-F) Oil red O staining of control cells (A), FFA-treated cells (B), cells treated with FFA+ S17902 (0.026 μM), (C) FFA+ S17902 (0.26 μM), (D) FFA+ S17902 (13 μM), (E) or FFA+ S17902 (26 μM) (F) for 24 h; (G) TG concentration in the L02 cells treated with S17092 for 24 h.(H) FASN/PPAR-γ/SREBP-1c mRNA expression levels in the controls and FFA-treated L02 cells with or without S17092 incubation for 24 h. (I-J) LC3B II protein expression levels in the controls and FFA-treated L02 cells with or without S17092 incubation for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Indeed, our results demonstrated that LC3B II, a bioprotein marker of autophagy, was significantly increased in the L02 cells that were treated with FFA for 24 h, which implies enhanced autophagy and is in agreement with a previous study [ ].

Techniques: Staining, Control, Concentration Assay, Expressing, Incubation