Journal: Scientific Reports

Article Title: Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

doi: 10.1038/s41598-023-49136-8

Figure Lengend Snippet: Overexpression of Gankyrin suppresses ferroptosis in TNBC cells. ( A ) Gankyrin protein expression in Hs578T and MDA-MB-231 cells transfected with shControl, shGankyrin #1, and shGankyrin #2 vectors, respectively. ( B ) Flow cytometry analysis of the proportion of 7-AAD-positive cells in Hs578T and MDA-MB-231 cells. ( C ) Fold changes in lactate dehydrogenase (LDH) release compared to the control. ( D ) Flow cytometry detection of lipid peroxides C11-BODIPY. ( E – G ) LDH release ( E ), proportion of 7-AAD-positive cells ( F ), and lipid peroxides C11-BODIPY ( G ) in Hs578T and MDA-MB-231 cells after gankyrin inhibition by cjoc42. Data represent the means ± standard deviations of three independent replicates and were analyzed using one-way univariate analysis of variance with multiple comparisons. Statistical significance was denoted as *** < 0.001 and **** < 0.0001.

Article Snippet: All the flow cytometry analyses were performed by the CyFlow Cube 6 system (Sysmex, Kobe, Japan) and then analyzed and images captured by FlowJo software (BD Biosciences).

Techniques: Over Expression, Expressing, Transfection, Flow Cytometry, Control, Inhibition

Journal: Scientific Reports

Article Title: Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

doi: 10.1038/s41598-023-49136-8

Figure Lengend Snippet: Effect of gankyrin on p53 protein expression in TNBC cells. ( A ) Levels of p53 protein expression in Hs578T and MDA-MB-231 cells after transfection with the designated siRNA interfering vectors. ( B ) Fluorescence intensity of p53 in Hs578T and MDA-MB-231 cells detected by Flow cytometry. ( C ) Expression of TP53 mRNA in Hs578T and MDA-MB-231 cells was detected by conventional RT-PCR. ( D ) Expression of TP53 mRNA in Hs578T and MDA-MB-231 cells was quantified by RT-qPCR. The results are presented as the mean ± standard deviation of three independent replicates and were analyzed using a one-way univariate analysis of variance with multiple comparisons. Statistical significance was determined as follows: ** < 0.01, *** < 0.001, **** < 0.0001, and ns indicating no statistical difference.

Article Snippet: All the flow cytometry analyses were performed by the CyFlow Cube 6 system (Sysmex, Kobe, Japan) and then analyzed and images captured by FlowJo software (BD Biosciences).

Techniques: Expressing, Transfection, Fluorescence, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

Journal: Scientific Reports

Article Title: Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

doi: 10.1038/s41598-023-49136-8

Figure Lengend Snippet: Involvement of p53 expression in the inhibition of ferroptosis. Hs578T and MDA-MB-231 cells treated with erastin were transfected with the shCtrl empty or shGankyrin expression vector for 24 h, followed by a 2-h incubation with or without the p53 inhibitor PFT-α (15 μM). ( A ) Fold changes in LDH release (vs. the shCtrl) were measured. ( B ) Proportion of 7-AAD positive cells in each group was analyzed by flow cytometry. ( C ) Lipid oxidates C11-BODIPY were analyzed by flow cytometry. The data presented represent the mean ± standard deviation of three independent replicates and were analyzed using one-way univariate analysis of variance with multiple comparisons. Statistical significance was determined as follows: ** < 0.01, *** < 0.001, **** < 0.0001, and “ns” indicating no statistical difference.

Article Snippet: All the flow cytometry analyses were performed by the CyFlow Cube 6 system (Sysmex, Kobe, Japan) and then analyzed and images captured by FlowJo software (BD Biosciences).

Techniques: Expressing, Inhibition, Transfection, Plasmid Preparation, Incubation, Flow Cytometry, Standard Deviation

Journal: Scientific Reports

Article Title: Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

doi: 10.1038/s41598-023-49136-8

Figure Lengend Snippet: Role of PSMD10 in inhibiting ferroptosis in TNBC cells through the p53/SLC7A11/GPX4 pathway. ( A ) Protein expression of p53, SLC7A11, GPX4, MDM2, and gankyrin were examined in HEK293T cells expressing MDM2 with a HA-MDM2 vector. ( B ) Ubiquitination level of p53 (left panel) and the protein expression levels of p53, SLC7A11, GPX4, and gankyrin were assessed in Hs578T cells (right panel). ( C ) Hs578T and MDA-MB-231 cells were transfected with shGankyrin or shCtrl, along with a HA-MDM2 plasmid transfection, and the relative RNA expression levels of TP53 and SLC7A11 were measured. ( D ) Fold change in cystine uptake level compared to the shCtrl was determined. ( E ) Relative RNA expression level of GPX4 compared to the shCtrl was examined by RT-qPCR. (F) Fold change in LDH release compared to the control was measured. ( G and H ) Percentage of 7-AAD-positive dead cells ( G ) and the fold change in lipid peroxide C11-BODIPY compared to the control ( H ) were analyzed by flow cytometry. The data represent the mean ± SD from three independent experiments and were analyzed using two-way ANOVA followed by Tukey’s post-hoc multiple comparison analysis. Statistical significance is denoted as * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. NS indicates no significance.

Article Snippet: All the flow cytometry analyses were performed by the CyFlow Cube 6 system (Sysmex, Kobe, Japan) and then analyzed and images captured by FlowJo software (BD Biosciences).

Techniques: Expressing, Plasmid Preparation, Ubiquitin Proteomics, Transfection, RNA Expression, Quantitative RT-PCR, Control, Flow Cytometry, Comparison