Journal: Cancers

Article Title: Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer

doi: 10.3390/cancers11091255

Figure Lengend Snippet: NRF2 antibody validation. ( A ) NRF2 expression was silenced in MCF7 cells by transiently transfecting NRF2-specific siRNAs or a negative control siRNA for 48 h, then NRF2 protein expression was determined using two different antibodies (Abcam: ab31163; NOVUS: NBP1-32822). ( B – D ) 4T1 cells were treated with NRF2 activators, RA839 or tBHQ, or MG-132, a proteasome inhibitor, in the concentrations indicated for 48 h, then NRF2 protein expression was determined by western blotting using two different antibodies (Abcam: ab31163; NOVUS: NBP1-32822).

Article Snippet: RA839, an NRF2 activator, which were obtained from Tocris Bioscience (Bristol, UK) and were used at a final concentrations of 5 μm and/or 10 μm.

Techniques: Biomarker Discovery, Expressing, Negative Control, Western Blot

Journal: Cancers

Article Title: Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer

doi: 10.3390/cancers11091255

Figure Lengend Snippet: LCA inhibited the NRF2/KEAP1 system. ( A , B ) The 4T1 cells were treated with LCA in the concentrations indicated for 48 h, then ( A ) NRF2 and ( B ) KEAP1 proteins were analyzed by western blotting. ( n = 3, upper panel: representative figure, lower panel: densitometric analysis of western blots from independent experiments). ( C ) The 4T1 cells were treated with 0.3 μm LCA and/or the NRF2 activator, RA839, in the concentrations indicated for 48 h, then total protein concentration was determined by sulforhodamine B assay ( n = 5). Data are plotted as mean ± SEM. * indicates p < 0.05, control vs. LCA-treated groups. (ns, not significant; KEAP1, Kelch-like ECH associating protein 1; LCA, lithocholic acid; NRF2, nuclear factor).

Article Snippet: RA839, an NRF2 activator, which were obtained from Tocris Bioscience (Bristol, UK) and were used at a final concentrations of 5 μm and/or 10 μm.

Techniques: Western Blot, Protein Concentration, Sulforhodamine B Assay, Control

Journal: Cancers

Article Title: Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer

doi: 10.3390/cancers11091255

Figure Lengend Snippet: Pharmacological activation of NRF2 induced the expression of NRF2 target genes. The 4T1 cells were treated with the NRF2 activator, RA839, in the concentrations indicated for 48 h, then the expressions of NRF2 target genes, NQO1 , GCLC , CAT , and HMOX1, were determined using RT-qPCR ( n = 3). Abbreviations: NAD(P)H quinone dehydrogenase 1 ( NQO1 ), glutamate–cysteine ligase catalytic subunit ( GCLC ), catalase ( CAT ), and heme oxygenase 1 ( HMOX1 ). Data are plotted as mean ± SD. ** and *** indicate statistically significant differences between control and RA839-treated groups at p < 0.01 or p < 0.001, respectively.

Article Snippet: RA839, an NRF2 activator, which were obtained from Tocris Bioscience (Bristol, UK) and were used at a final concentrations of 5 μm and/or 10 μm.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control

Journal: Cancers

Article Title: Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer

doi: 10.3390/cancers11091255

Figure Lengend Snippet: NRF2 activation modulated LCA-induced oxidative stress responses in 4T1 breast cancer cells. The 4T1 cells were treated with 0.3 μm LCA and the NRF2 activator RA839 or tBHQ in the concentrations indicated for 48 h. Lipid peroxidation was determined by measuring ( A ) TBARS ( n = 4) and ( B , C ) 4-HNE levels using western blotting ( n = 3). ( D ) The 4T1 cells were treated with LCA (0.3 μm) and/or GSH and NAC (both at 5 mm) antioxidants for 48 h, then total protein concentration was determined using the sulforhodamine B assay ( n = 3). For statistical analysis ANOVA test was used followed by the Dunnett post-hoc test, where all groups were compared to the LCA-treated cohort. Data are plotted as mean ± SEM. ** p < 0.01, LCA vs. LCA/NRF2-activator-treated groups (GSH, reduced glutathione; LCA, lithocholic acid; NAC, N-acetylcysteine; TBARS, thiobarbituric acid reactive substances; 4HNE, 4-hydroxynonenal).

Article Snippet: RA839, an NRF2 activator, which were obtained from Tocris Bioscience (Bristol, UK) and were used at a final concentrations of 5 μm and/or 10 μm.

Techniques: Activation Assay, Western Blot, Protein Concentration, Sulforhodamine B Assay