Journal: Cell Death & Disease

Article Title: Gambogic acid triggers vacuolization-associated cell death in cancer cells via disruption of thiol proteostasis

doi: 10.1038/s41419-019-1360-4

Figure Lengend Snippet: a , c Cells were treated with the indicated concentrations of GA for 24 h. Cellular viability was assessed using IncuCyte as described in Materials and Methods. Data represent the means ± SEM ( n = 3). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.01 vs. untreated control. b , d Cells treated with GA at the concentrations around the IC 50 , (MDA-MB 453 (1.5 μM), MDA-MB 468 (2.35 μM), MDA-MB 435S (1.33 μM), BxPC-3 (1.68 μM), NCI-H460 (2.35 μM), SNU-449 (1.62 μM), and SNU-668 (1.41 μM)), which were calculated using GraphPad Prism, were observed by phase-contrast microscopy. MCF-10A cells treated with GA at the indicated concentrations for 24 h were observed by phase-contrast microscopy. Bars, 40 μm. e Athymic nude mice of 6–8 weeks old were xenografted with MDA-MB 435S cells and injected with vehicle, 4 mg/kg GA, and 8 mg/kg GA as described in Materials and Methods. Tumor sizes were measured every 2–3 days after the beginning of vehicle or GA injection and plotted for growth curve. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. * p < 0.05 vs. vehicle-treated mice. f treated mice and tumors isolated from those mice were photographed on the 14th day. g The results of H&E staining in tumor tissues of the mice treated with 4 mg/kg GA. Bars, 20 μm

Article Snippet: The plates were imaged on an IncuCyte device (Essen Bioscience, Ann Arbor, MI, USA) and analyzed using the IncuCyte ZOOM 2016B software.

Techniques: Microscopy, Injection, Isolation, Staining

Journal: Cell Death & Disease

Article Title: Gambogic acid triggers vacuolization-associated cell death in cancer cells via disruption of thiol proteostasis

doi: 10.1038/s41419-019-1360-4

Figure Lengend Snippet: a Proposed chemical structures of the GA-GSH and GA-NAC adducts. b Full-scan product ion scan spectra and the expected structures of GA, GA-GSH, and GA-NAC adduct formed upon Michael addition of GSH or NAC. The m/z values of the GA-GSH adduct represent GSH at 308, GA at 629, and the adduct form at 936. The m/z values of the GA-NAC adduct represent NAC at 164, GA at 651, and the adduct form at 814. c Increasing concentrations of NAC were pre-incubated with 1 μM GA in serum-free medium for the indicated time durations at room temperature, and these mixtures were used to treat MDA-MB 435S cells for 24 h. The cell viability was measured using IncuCyte. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. * p < 0.05; ** p < 0.01 vs. GA-treated cells. d MDA-MB 435S cells were treated with the indicated concentrations of GA or 10 μM iodoacetamide (IAM; a positive control to reduce intracellular protein-SH levels) for 12 h. Protein-SH levels were measured using the dibromobimane (dBrB) assay, as described in the Materials and Methods. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. * p < 0.01 vs. untreated control. e Cell extracts were prepared from the cells pretreated with the antioxidants and further treated with GA (1 μM for MDA-MB 435S and 2 μM for MDA-MB 453 cells) for the indicated concentrations for Western blotting. β-actin was used as a loading control. f MDA-MB 435S cells pretreated with 2 mM NAC and further treated with 1 μM GA for the indicated time points were incubated with TMRM. Samples were subjected for flow cytometry

Article Snippet: The plates were imaged on an IncuCyte device (Essen Bioscience, Ann Arbor, MI, USA) and analyzed using the IncuCyte ZOOM 2016B software.

Techniques: Incubation, Positive Control, Western Blot, Flow Cytometry