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: Scientific Reports

Article Title: Immune cell diversity and regenerative markers reveal interactions among macrophages, rodlet cells, and stem cells in the kidney of Poecilia sphenops

doi: 10.1038/s41598-025-11679-3

Figure Lengend Snippet: Immunohistochemistry of Nrf2 and Sox9. ( A , B ) Stem cells (arrowheads) around the blood vessels (BV) expressed Nrf2. ( C , D ) Stem cells (arrowheads) expressed Sox9.

Article Snippet: The sections were exposed to diluted (1:100) primary antibodies against the rabbit polyclonal S100 protein for an entire night at 4 °C (Z0311, Dako, Glostrup, Denmark), mouse monoclonal anti-CD68 (Santa Cruz, sc-17832), rabbit polyclonal Nicotinic Acetylcholine R alpha 7 NACHRA7 (ABclonal, A7844), rabbit polyclonal interleukin 1 beta (IL-1β) (sc-7884, Santa Cruz Biotechnology, Heidelberg Germany), rabbit polyclonal iNOS-2 (RB-1605, Thermo Fisher Scientific, UK), mouse monoclonal autophagy protein 5 (APG5) (sc-133158, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit polyclonal nuclear factor kappa B (NF-κB) (10745-1-AP, Proteintech, USA), rabbit polyclonal nuclear factor erythroid 2-related factor 2 (Nrf2) (sc-722, Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal SRY-Box transcription factor 9 (Sox9) (AB5535, Sigma-Aldrich, Madrid, Spain).

Techniques: Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: Hydrogen sulfide reduces oxidative stress in Huntington’s disease via Nrf2

doi: 10.4103/NRR.NRR-D-23-01051

Figure Lengend Snippet: Reagents and instruments used in the study

Article Snippet: Rabbit polyclonal anti-NRF2 antibody , Proteintech Group , 16396-1-AP (RRID: AB_2782956).

Techniques: CCK-8 Assay, Saline, TUNEL Assay, DNA Extraction, Modification, Marker, Fluorescence, Microscopy, Imaging

Journal: Neural Regeneration Research

Article Title: Hydrogen sulfide reduces oxidative stress in Huntington’s disease via Nrf2

doi: 10.4103/NRR.NRR-D-23-01051

Figure Lengend Snippet: NaHS reduces oxidative stress by suppressing Nrf2. (A) Representative images of DCFHDA staining in PC12 cells treated with Quin for 24 hours. The Quin group exhibited greater DCFH-DA (green) fluorescence than the Sham group. The Quin + NaHS group exhibited a decrease in DCFHDA (green) fluorescence compared with the Quin group, while an increase was observed in the Quin + NaHS + ML385 group. Scale bar: 50 μm. (B) DHE staining of PC12 cells following 24-hour Quin stimulation. Stronger DHE (red) fluorescence was observed in the Quin group compared with the Sham group. DHE (red) fluorescence was decreased in the Quin + NaHS group and increased in the Quin + NaHS + ML385 group compared with the Quin group. Scale bar: 50 μm. (C) DCFH-DA fluorescence intensity in PC12 cells was quantified using a fluorescence microplate reader ( n = 5 per group). (D) DHE fluorescence intensity in PC12 cells was quantified using a fluorescence microplate reader ( n = 5 per group). (E) DCFHDA fluorescence intensity in the striatum was quantified using a fluorescence microplate reader ( n = 5 per group). (F) Western blot detection of Nrf2 and HO-1 expression levels in PC12 cells in each group. Quantification of Nrf2 and HO-1 expression levels ( n = 3 per group). (G) Western blot detection of Nrf2 and HO-1 expression levels in the striatum of mice in each group. Quantification of Nrf2 and HO-1 expression levels ( n = 3 per group). The values are reported as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Bonferroni post hoc test). DCFHDA: 2′,7′-Dichlorodihydrofluorescein diacetate; DHE: dihydroethidium; HO-1: heme oxygenase-1; Nrf2: nuclear factor erythroid 2-related factor 2; Quin: quinolinic acid.

Article Snippet: Rabbit polyclonal anti-NRF2 antibody , Proteintech Group , 16396-1-AP (RRID: AB_2782956).

Techniques: Staining, Fluorescence, Western Blot, Expressing

Journal: Neural Regeneration Research

Article Title: Hydrogen sulfide reduces oxidative stress in Huntington’s disease via Nrf2

doi: 10.4103/NRR.NRR-D-23-01051

Figure Lengend Snippet: NaHS regulates mitochondrial function in Quin-stimulated PC12 cells by suppressing Nrf2. (A) Mitochondria stained with MitoTracker (red) in Quin-stimulated PC12 cells treated with NaHS and ML385. The Quin group exhibited severe mitochondrial fragmentation and a decrease in mitochondrial fluorescence intensity compared with the Sham group, whereas the addition of NaHS resulted in an increase in mitochondrial fluorescence intensity. However, this effect was reversed by the addition of ML385. Scale bar: 10 μm. (B) Quantification of mitochondrial fluorescence intensity ( n = 10 per group). (C) Representative micrographs of MMP in Quin-stimulated PC12 cells, as detected via JC-1. The transition from green to red fluorescence indicates mitochondrial polarization, which was restored to normal levels by NaHS treatment. In contrast, ML385 treatment induced a shift from green to red fluorescence, indicating substantial mitochondrial depolarization. Scale bar: 50 μm. (D) Quantification of mitochondrial MMP ( n = 15 per group). (E) Quantification of mitochondrial DNA (mtDNA, NADH5) versus nuclear DNA (GAPDH) as detected by real-time polymerase chain reaction in Quin-stimulated PC12 cells treated with NaHS and ML385. (F) Western blot detection of TOMM20 expression levels in PC12 cells. Quantification of TOMM20 expression ( n = 3 per group). The values are reported as mean ± SD. ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Bonferroni post hoc test). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; MMP: mitochondrial membrane potential; Quin: quinolinic acid; TOMM20: translocase of outer mitochondrial membrane 20.

Article Snippet: Rabbit polyclonal anti-NRF2 antibody , Proteintech Group , 16396-1-AP (RRID: AB_2782956).

Techniques: Staining, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Membrane