Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: CNE1 cells were treated with or without CuB (1, 10, 50, 100,200,500 and 1000 nM) for 48 h. Then, the cells were harvested, stained with Annexin V-FITC/ PI solution for flow cytometer analysis ( A ), or lysed to harvest the total proteins for the western blot analysis of caspase-3, cleaved caspase-3, PARP, and cleaved PARP. ** p < 0.01 and *** p < 0.001 vs . the control group by t -test using GraphPad Prism 5.0. B A minimum of 10,000 cells were collected for analysis by flow cytometer (Navios, Beckman Coulter). Quantitative analysis was performed by Image J (NIH, MD, USA). Data were presented as the mean ± SEM of three independent experiments. ** p < 0.01 and *** p < 0.001 vs . the control group by t -test using GraphPad Prism 5.0.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: A The cytotoxicity of CuB at the concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 nM towards CNE1 cells with or without Nec1 (50 μM), ZVAD (50 μM), Fer-1 (5 μM), CPX (5 μM), or DFO (100 μM) for 48 h, respectively, and the cytotoxicity were determined by MTT assay. B The LDH released in CNE1 cells exposed to CuB at the concentrations of 1, 10, and 50 nM with or without Nec1(50 μM), ZVAD(50 μM), Fer-1(5 μM), CPX (5 μM), or DFO (100 μM) for 24 h, respectively. *** p < 0.001 vs . the CuB-treated group by t-test using GraphPad Prism 5.0. C Representative phase-contrast images of the CNE1 cells exposed to CuB at 50 nM for 24 h ( b ) and 48 h ( d ) with or without Nec1(50 μM, e – h ), ZVAD(50 μM, i – l ), CPX (5 μM, m – p ), DFO (100 μM, q – t ) or Fer-1(5 μM, u – x ) by electron microscope (Nikon, Japan). Scar bar: 20 μm. Data were presented as the mean ± SEM of three independent experiments.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: MTT Assay, Microscopy

Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: A Representative cell and mitochondrial ultrastructural images of CNE1 cells treated with or without CuB (50 nM) or erastin (10 μΜ) for 24 h. The cells were fixed and detected by TEM (HT7700, Hitachi, Ibaraki, Japan). Scale bars: cell, 5 μm; mitochondria, 500 nm. B Intracellular iron content in CNE1 cells exposed to CuB (1, 10, and 50 nM) for 24 h was determined with an iron assay kit (Sigma, USA). ** p < 0.01 vs . the control group by t -test using GraphPad Prism 5.0. C Intracellular GSH content in CNE1 cells exposed to CuB (1,10 and 50 nM) for 6 h, 12 h, or 24 h was determined with a GSH assay kit (Beyotime, Jiangsu, China). *** p < 0.001 vs . the control group by t -test using GraphPad Prism 5.0. D The lipid peroxidation generation in CNE1 cells exposed to CuB (1, 10 and 50 nM) with or without Nec1(50 μM), ZVAD(50 μM), Fer-1(5 μM), CPX (5 μM), or DFO (100 μM) for 24 h. Then harvested cells were stained with C 11 -BODIPY581/591 and analysed by flow cytometer (Navios, Beckman Coulter). A minimum of 10,000 cells were harvested for analysis. [MFI = mean fluorescence intensity]. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs . control group by t -test using GraphPad Prism 5.0. E The fluorescent images of lipid peroxidation in CNE1 cells using C 11 -BODIPY-581/591 probe after incubation with CuB (1, 10, and 50 nM) or erastin (10 μM) for 24 h. Scale bar: 20 μm. ( F ) The mRNA level of GPX4 quantified by qRT-PCR and western blot analysis of GPX4 in CNE1 cells with or without CuB (1, 10, and 50 nM) for 24 h. Quantitative analysis was performed by Image J (NIH, MD, USA). *** p < 0.001 vs . control group by t -test using GraphPad Prism 5.0. Data were presented as the mean ± SEM of three independent experiments.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: Iron Assay, GSH Assay, Staining, Flow Cytometry, Fluorescence, Incubation, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: A Representative images of intracellular microtubule networks of CNE1 cells treated with or without CuB (50 nM) 24 h, and then direct detected ( a and g ) by confocal laser scanning microscope (Leica Microsystems, Germany). In the microtubules reassembly assay, the untreated or treated cells were placed on ice for 1 h, subsequently warm at 37 °C for 0 ( b and h ), 3 ( c and i ), 6 ( d and j ), 9 ( e and k ) and 15 min ( f and l ) and then observed by confocal microscopy. The microtubules were stained with β-tubulin mouse antibody and Dylight™ 488-Conjugated Goat Anti-Mouse IgG secondary antibody. The nucleus was stained by Hoechst33342. Scale bars: 10 μm. ( B-C ) The effect of CuB on cell cycle arrest in CNE1 cells. CNE1 cells were treated with or without CuB at 10, 50, and 100 nM for 24 h, and then harvested cells were analysed for PI-stained DNA content by flow cytometry ( B ) or lysed for western blot analysis of cyclinB1, cdc25C, cdc2, and p-cdc2 ( C ). A minimum of 10,000 cells were harvested and analysed by EXPO32 ADC software. Quantitative analysis of western blots was performed by Image J (NIH, MD, USA). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs . control group by t -test using GraphPad Prism 5.0. Data were presented as the mean ± SEM of three independent experiments.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: Laser-Scanning Microscopy, Confocal Microscopy, Staining, Flow Cytometry, Western Blot, Software

Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: A , B After 24 h of incubation with CuB (10, 50, and 100 nM), migratory and invasive behaviours were analysed using migration and matrigel invasion assays in CNE1 cells. *** p < 0.001 vs . control group by t -test using GraphPad Prism 5.0. Scale bars: 100 μm. C Western blot analysis of expressions of slug, β-catenin, E-cadherin, N-cadherin, snail, and vimentin in CNE1 cells with CuB (10, 50, and 100 nM) treatment for 48 h. Quantitative analysis of western blots was performed by Image J (NIH, MD, USA). ** p < 0.01 and *** p < 0.001 vs . the control group by t -test using GraphPad Prism 5.0. Data were presented as the mean ± SEM of three independent experiments.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: Incubation, Migration, Western Blot

Journal: Cell Death & Disease

Article Title: Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

doi: 10.1038/s41419-021-03516-y

Figure Lengend Snippet: A Images of euthanized mice with or without the administration of CuB (0.5, 1 mg/kg) and GEM (25 mg/kg). B Images of excised tumour bulks and final average tumour volume in each group. C Body weight of mice from each group during the whole observation period. Data were presented as the mean ± SEM, * p < 0.05 vs. the control group by t -test using GraphPad Prism 5.0, n = 5. # p < 0.05 for between group comparisons by one-way ANOVA using GraphPad Prism 5.0, n = 5.

Article Snippet: The viability of cells treated with the compounds was evaluated by calculating the IC 50 values with GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA).

Techniques: