Journal: Redox Biology

Article Title: Beyond CFTR: Ivacaftor's role in restoring cellular redox balance and preventing ferroptosis

doi: 10.1016/j.redox.2025.103944

Figure Lengend Snippet: Ivacaftor reduces lipid peroxidation and protects CFBE cells from erastin-induced ferroptosis. A-B. The bar graphs show the ratio FITC-A/PE-A mean fluorescence intensity (oxidized/reduced) C11-BODIPY. Error bars are mean ± SD. A. CFBE cells manifest higher levels of lipid hydroperoxides compared to 16HBE after 24h in a 12-well plate. Paired Student's t -test (∗p < 0.05). B. CFBE cells were treated 24, 48, or 72h with elexacaftor (VX-445), tezacaftor (VX-661), lumacaftor (VX-809) or ivacaftor (VX-770) at the indicated concentrations. Vehicle DMSO was included as negative control, while RTA-408 as positive control. Data are expressed as fold change to DMSO. Ivacaftor reduces lipid hydroperoxides level in CFBE cells already after 24h of treatment. Ordinary one-way ANOVA multiple comparison vs. DMSO with Dunnett's correction (∗∗p < 0.01; ∗∗∗p < 0.001). C -D. The bar graphs show resazurin-based cell viability of CFBE cells, expressed as percentage relative to vehicle DMSO. Cells were treated with erastin (Era) alone or in combination (blue dots). Ordinary one-way ANOVA multiple comparison vs. Era with Dunnett's correction (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). C. VX-770 protects CFBE cells from Era-induced ferroptosis already at 24h and this effect persists over the 72h treatment. The Nrf2 activator RTA-408 does not protect from Era like vehicle DMSO, while ferroptosis inhibitor Fer-1 was included as a positive control. D. At 24h VX-770's protective ability in CFBE cells is maintained also when combined with CFTR correctors and in the triple-combination VX-770, VX-445 and VX-661 (ETI).

Article Snippet: Rabbit anti-xCT polyclonal antibody 1:1000 (26864-1-AP, Proteintech) Rabbit anti-Nrf2 polyclonal antibody 1:1000 (16396-1-AP, Proteintech) , Goat anti-rabbit IgG HRP conjugate, 1:5000 (NEF812001EA, Perkin Elmer).

Techniques: Fluorescence, Negative Control, Positive Control, Comparison

Journal: Redox Biology

Article Title: Beyond CFTR: Ivacaftor's role in restoring cellular redox balance and preventing ferroptosis

doi: 10.1016/j.redox.2025.103944

Figure Lengend Snippet: Ivacaftor modulates the Nrf2-based antioxidant response in CFBE cells. A. Representative Western blot analysis of Nrf2 protein. CFBE cells display a decreased level of Nrf2 compared to 16HBE cells. Values in the bar graph represent normalized integrated density of Western blot, expressed as fold change to 16HBE. Error bars are mean ± SD. Unpaired Student's t -test (∗∗p < 0.01). Representative Western blot of Nrf2 (B) or xCT (C) proteins in CFBE cells treated 24, 48, or 72h with ivacaftor (VX-770) 10 μM. Vehicle DMSO was used as control. Filled dots correspond to addition of the compound/vehicle. Values in the bar graphs represent normalized integrated density of Western blot and are expressed as fold change to DMSO. Error bars are mean ± SD. Unpaired Student's t -test (∗p < 0.05; ∗∗∗p < 0.001). VX-770 significantly increases Nrf2 level at 24h (B) and xCT at 48h (C) . D. 72-h treatment with VX-770 10 μM significantly increases total glutathione (tGSH) in CFBE cells. RTA-408 was included as positive control. tGSH was measured with Tietze assay and normalized to total intracellular proteins. Each point represents an independent biological replicate. Error bars are mean ± SD. Ordinary one-way ANOVA multiple comparison vs. DMSO with Dunnett's correction (∗p < 0.05; ∗∗p < 0.01). E. Similarly to the Nrf2 activator RTA-408 included as positive control, VX-770 10 μM increases the normalized relative luciferase activity in CFBE cells transiently transfected with the pGL3-8xARE-luciferase Nrf2 reporter plasmid. Each point represents an independent biological replicate and is expressed as fold change to DMSO. Error bars are mean ± SD. Ordinary one-way ANOVA multiple comparison vs. DMSO with Dunnett's correction (∗p < 0.05; ∗∗p < 0.01).

Article Snippet: Rabbit anti-xCT polyclonal antibody 1:1000 (26864-1-AP, Proteintech) Rabbit anti-Nrf2 polyclonal antibody 1:1000 (16396-1-AP, Proteintech) , Goat anti-rabbit IgG HRP conjugate, 1:5000 (NEF812001EA, Perkin Elmer).

Techniques: Western Blot, Control, Positive Control, Comparison, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Redox Biology

Article Title: Beyond CFTR: Ivacaftor's role in restoring cellular redox balance and preventing ferroptosis

doi: 10.1016/j.redox.2025.103944

Figure Lengend Snippet: Ivacaftor's regulates FSP1 protein level but does not base its anti-ferroptotic activity on it. A. Representative Western blot of FSP1 protein in CFBE cells treated 24, 48, or 72h with ivacaftor (VX-770) 10 μM. Vehicle DMSO was used as control. Filled dots correspond to addition of the compound/vehicle. Values in the bar graphs represent normalized integrated density of Western blot and are expressed as fold change to DMSO. Error bars are mean ± SD. Unpaired Student's t -test (∗p < 0.05). VX-770 significantly increases FSP1 level at 24h. Western blot validation of the CRISPR-Cas9-generated CFBE Nrf2 KO (B) and FSP1 KO (C) cells, compared to the non-target control (N.T.) CFBE cell line. D. Resazurin-based cell viability assay of N.T., Nrf2 KO and FSP1 KO CFBE cells treated 24h with the ferroptosis inducer erastin. Concentrations of erastin varied in a range between 0 and 10 μM. VX-770 5 μM efficiently protected each cell line from erastin-induced ferroptosis, thus suggesting that VX-770's activity does not depend on Nrf2 and FSP1. Each point represents the mean ± SD of at least three independent biological replicates.

Article Snippet: Rabbit anti-xCT polyclonal antibody 1:1000 (26864-1-AP, Proteintech) Rabbit anti-Nrf2 polyclonal antibody 1:1000 (16396-1-AP, Proteintech) , Goat anti-rabbit IgG HRP conjugate, 1:5000 (NEF812001EA, Perkin Elmer).

Techniques: Activity Assay, Western Blot, Control, Biomarker Discovery, CRISPR, Generated, Viability Assay

Journal: Food Science & Nutrition

Article Title: Trans‐Fatty Acids ( TFA ) Induced Vascular Injury Through the Regulation of the Sirt1‐Ppargc1a‐Nfe2l2 Signaling Pathway in Male Rats

doi: 10.1002/fsn3.70975

Figure Lengend Snippet: Effect of trans‐fatty acids on Sirt1/Ppargc1a/Nfe2l2 pathway of rat blood vessels. (A) Western blotting results of Silent information regulator 1 (Sirt1), Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (Ppargc1a), and nuclear factor erythroid 2‐related factor 2 (Nfe2l2). (B−D) Semi‐quantitative analyses of protein expression. Results are shown as mean ± SD ( n = 6). * p < 0.05 by contrast with ND group; # p < 0.05 by contrast with LTD group. ND, normal diet; LTD, 1% trans‐fatty acids diet; HTD, 4% trans‐fatty acids diet.

Article Snippet: The following primary antibodies were employed in the study: anti‐rabbit Bcl2 (Signalway Antibody, China), anti‐rabbit Bax (Cell Signaling Technology, USA), anti‐rabbit Ppargc1a (Affinity, China), anti‐rabbit Casp3 (GeneTex, USA), anti‐rabbit Cleaved Casp3 (Arigo Biolaboratories, China), anti‐rabbit Nfe2l2 (Medical & Biological Laboratories, China), anti‐rabbit Sirt1 (OriGene, USA), and anti‐rabbit β‐actin (Abcam, China).

Techniques: Western Blot, Expressing