Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis

doi: 10.3390/ani12213026

Figure Lengend Snippet: List of primers employed for q-RT-PCR.

Article Snippet: The proteins were then transferred onto PVDF membranes (GE Bioscience, Newark, NJ, USA) and then blocked with 5% BSA (dissolved in TBST) for 1 h and the membrane was incubated with anti-SYCP3 (ab97672; Abcam, Cambridge, UK), anti-DDX4 (bs-22896R; Bioss Biotech, Beijing, China), anti-SOX9 (ab185966; Abcam, Cambridge, UK), anti-SLC7A11 (bs-6883R; Bioss Biotech, Beijing, China), anti-GPX4 (bs-3884R; Bioss Biotech, Beijing, China), anti-4-HNE (ab48506; Abcam), and anti-β-actin (bs-0061R; Bioss Biotech, Beijing, China) overnight at 4 °C.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis

doi: 10.3390/ani12213026

Figure Lengend Snippet: Expressions of genes, proteins involved in ferroptosis in testis of mice in each group. ( A ) Nrf2 mRNA expression. ( B ) SLC7A11 mRNA expression. ( C ) GPX4 mRNA expression. ( D ) Western blot detection of SLC7A11, GPX4, and 4-HNE protein. ( E ) Image J analysis showing the grey value of SLC7A11. ( F ) Image J analysis showing the grey value of GPX4. ( G ) Image J analysis showing the grey value of 4-HNE. The data are expressed as means ± SEM, with n ≥ 3; Different lowercase letters (a, b, c, d) indicate significant differences ( p < 0.05).

Article Snippet: The proteins were then transferred onto PVDF membranes (GE Bioscience, Newark, NJ, USA) and then blocked with 5% BSA (dissolved in TBST) for 1 h and the membrane was incubated with anti-SYCP3 (ab97672; Abcam, Cambridge, UK), anti-DDX4 (bs-22896R; Bioss Biotech, Beijing, China), anti-SOX9 (ab185966; Abcam, Cambridge, UK), anti-SLC7A11 (bs-6883R; Bioss Biotech, Beijing, China), anti-GPX4 (bs-3884R; Bioss Biotech, Beijing, China), anti-4-HNE (ab48506; Abcam), and anti-β-actin (bs-0061R; Bioss Biotech, Beijing, China) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Experimental and therapeutic medicine

Article Title: Novel function of fluvastatin in attenuating oxidized low-density lipoprotein-induced endothelial cell ferroptosis in a glutathione peroxidase4- and cystine-glutamate antiporter-dependent manner.

doi: 10.3892/etm.2021.10710

Figure Lengend Snippet: Figure 1. ox‑LDL promotes endothelial cell dysfunction via induction of ferroptosis. (A) Immunoblots of TNF‑α, IL‑α, 4‑HNE, GPx4 and xCT in the normal (control), ox‑LDL and ox‑LDL + DFOM groups. (B) Representative Transwell migration assay images of HUVECs treated with ox‑LDL and ox‑LDL + DFOM (magnification, x10). (C) Representative tube formation assay images of HUVECs treated with PBS, 100 µg/ml ox‑LDL and 125 µM DFOM (magnification, x10). (D) Analysis of HUVEC viability using the MTT assay after ox‑LDL treatment. Columns, means of three experiments; bars, SD. Histograms of (E) relative TNF‑α/β‑actin levels, (F) relative IL‑α/β‑actin levels, (G) relative 4‑HNE/β‑actin levels, (H) relative GPx4/β‑actin levels and (I) relative xCT/β‑actin levels. *P<0.05 vs. control group. ox‑LDL, oxidized low‑density lipoprotein; GPx4, glutathione peroxidase 4; xCT, cystine‑glutamate antiporter; 4‑HNE, 4‑hydroxynonenal; DFOM, deferoxamine mesylate; HUVEC, human umbilical vein cell.

Article Snippet: The following primary antibodies were used: 4‐hydroxynonenal (4‐HNE; 1:2,000; ab46544; Abcam), TNF‐α (1:500; WL01581; Wanleibio; Co., Ltd.), IL‐1α (1:2,000; ab134908; Abcam), GPx4 (1:1,000; 14432‐1‐AP; ProteinTech Group, Inc.), xCT (1:1,000; 26864‐1‐AP; ProteinTech Group, Inc.); Caspase‐3/cleaved‐caspase3 (1:500; WL02117; Wanleibio; Co., Ltd.), Bax (1:1,000; WL01637; Wanleibio; Co., Ltd.), Bcl‐2 (1:1,000; WL01556; Wanleibio; Co., Ltd.), β‐actin (1:2,000; WL01845 Wanleibio; Co., Ltd.).

Techniques: Western Blot, Control, Transwell Migration Assay, Tube Formation Assay, MTT Assay

Journal: Experimental and therapeutic medicine

Article Title: Novel function of fluvastatin in attenuating oxidized low-density lipoprotein-induced endothelial cell ferroptosis in a glutathione peroxidase4- and cystine-glutamate antiporter-dependent manner.

doi: 10.3892/etm.2021.10710

Figure Lengend Snippet: Figure 2. RNAi‑mediated GPx4 and xCT knockdown promotes ox‑LDL‑induced ferroptosis in human endothelial cells. (A) Immunoblots of TNF‑α, IL‑α, 4‑HNE, GPx4 and xCT in the normal (control), ox‑LDL, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + DFOM, si‑xCT + ox‑LDL and si‑xCT + ox‑LDL + DFOM treatment groups. (B) Representative Transwell migration assay images of HUVECs treated with ox‑LDL, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + DFOM, si‑xCT + ox‑LDL and si‑xCT + ox‑LDL + DFOM (magnification, x10). #P<0.05 vs. control group; *P<0.05 vs. si‑GPx4 + ox‑LDL; -P<0.05 vs. si‑xCT + ox‑LDL. (C) Representative images showing HUVEC tube formation in the presence of PBS (control) or 100 µg/ml ox‑LDL,si‑GPx4 + 100 µg/ml ox‑LDL, si‑GPx4 + 100 µg/ml ox‑LDL + 125 µM DFOM, si‑xCT + 100 µg/ml ox‑LDL or si‑xCT + 100 µg/ml ox‑LDL + 125 µM DFOM (all magnifications, x10). #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL, ‑P<0.05 vs. si‑xCT + ox‑LDL. (D) Analysis of cell viability using the MTT assay in HUVECs after ox‑LDL, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + DFOM, si‑xCT + ox‑LDL and si‑xCT + ox‑LDL + DFOM treatments. Columns, mean of three experiments; bars, SD. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL, ‑P<0.05 vs. si‑xCT + ox‑LDL. (E) Histogram of relative TNF‑α/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL. (F) Histogram of relative IL‑α/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL. (G) Histogram of relative 4‑HNE/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL. (H) Histogram of relative xCT/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL. (I) Histogram of relative GPx4/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑GPx4 + ox‑LDL. (J) Histogram of relative TNF‑α/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑xCT + ox‑LDL. (K) Histogram of relative IL‑α/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑xCT + ox‑LDL. (L) Histogram of relative 4‑HNE/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑xCT + ox‑LDL. (M) Histogram of relative GPx4/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑xCT + ox‑LDL. (N) Histogram of relative xCT/β‑actin levels. #P<0.05 vs. control group, *P<0.05 vs. si‑xCT + ox‑LDL. ox‑LDL, oxidized low‑density lipoprotein; GPx4, glutathione peroxidase 4; xCT, cystine‑glutamate antiporter; 4‑HNE, 4‑hydroxynonenal; DFOM, deferoxamine mesylate; HUVEC, human umbilical vein cell.

Article Snippet: The following primary antibodies were used: 4‐hydroxynonenal (4‐HNE; 1:2,000; ab46544; Abcam), TNF‐α (1:500; WL01581; Wanleibio; Co., Ltd.), IL‐1α (1:2,000; ab134908; Abcam), GPx4 (1:1,000; 14432‐1‐AP; ProteinTech Group, Inc.), xCT (1:1,000; 26864‐1‐AP; ProteinTech Group, Inc.); Caspase‐3/cleaved‐caspase3 (1:500; WL02117; Wanleibio; Co., Ltd.), Bax (1:1,000; WL01637; Wanleibio; Co., Ltd.), Bcl‐2 (1:1,000; WL01556; Wanleibio; Co., Ltd.), β‐actin (1:2,000; WL01845 Wanleibio; Co., Ltd.).

Techniques: Knockdown, Western Blot, Control, Transwell Migration Assay, MTT Assay

Journal: Experimental and therapeutic medicine

Article Title: Novel function of fluvastatin in attenuating oxidized low-density lipoprotein-induced endothelial cell ferroptosis in a glutathione peroxidase4- and cystine-glutamate antiporter-dependent manner.

doi: 10.3892/etm.2021.10710

Figure Lengend Snippet: Figure 3. Fluvastatin attenuates ox‑LDL‑induced endothelial cell dysfunction and ferroptosis. (A) Immunoblots of TNF‑α, IL‑α, 4‑HNE, GPx4 and xCT in the normal, ox‑LDL, ox‑LDL + 2.5 µM fluvastatin, ox‑LDL + 5 µM fluvastatin, ox‑LDL + 10 µM fluvastatin treatment groups. (B) Analysis of cell viability using the MTT assay in HUVECs after ox‑LDL, ox‑LDL + 2.5 µM fluvastatin, ox‑LDL + 5 µM fluvastatin and ox‑LDL + 10 µM fluvastatin treatments. Columns, means of three experiments; bars, SD. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (C) Histogram of relative TNF‑α/β‑actin levels. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (D) Histogram of relative IL‑α/β‑actin levels. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (E) Histogram of relative 4‑HNE/β‑actin levels. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (F) Histogram of relative GPx4/β‑actin levels. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (G) Histogram of relative xCT/β‑actin levels.*P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. (H) Representative Transwell migration assay images of HUVECs treated with ox‑LDL, ox‑LDL + 2.5 µM fluvastatin, ox‑LDL + 5 µM fluvastatin, or ox‑LDL + 10 µM fluvastatin. *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group (magnification, 100x). (I) Representative tube formation assay images of HUVEC in the presence of PBS (control) or with 100 µg/ml ox‑LDL, 100 µg/ml ox‑LDL + 2.5 µM fluvastatin, 100 µg/ml ox‑LDL + 5 µM fluvastatin, or 100 µg/ml ox‑LDL + 10 µM fluvastatin (magnification, 100x). *P<0.05 vs. control group, ΔP<0.05 vs. ox‑LDL group. ox‑LDL, oxidized low‑density lipoprotein; GPx4, glutathione peroxidase 4; xCT, cystine‑glutamate antiporter; 4‑HNE, 4‑hydroxynonenal; HUVEC, human umbilical vein cell.

Article Snippet: The following primary antibodies were used: 4‐hydroxynonenal (4‐HNE; 1:2,000; ab46544; Abcam), TNF‐α (1:500; WL01581; Wanleibio; Co., Ltd.), IL‐1α (1:2,000; ab134908; Abcam), GPx4 (1:1,000; 14432‐1‐AP; ProteinTech Group, Inc.), xCT (1:1,000; 26864‐1‐AP; ProteinTech Group, Inc.); Caspase‐3/cleaved‐caspase3 (1:500; WL02117; Wanleibio; Co., Ltd.), Bax (1:1,000; WL01637; Wanleibio; Co., Ltd.), Bcl‐2 (1:1,000; WL01556; Wanleibio; Co., Ltd.), β‐actin (1:2,000; WL01845 Wanleibio; Co., Ltd.).

Techniques: Western Blot, MTT Assay, Control, Transwell Migration Assay, Tube Formation Assay

Journal: Experimental and therapeutic medicine

Article Title: Novel function of fluvastatin in attenuating oxidized low-density lipoprotein-induced endothelial cell ferroptosis in a glutathione peroxidase4- and cystine-glutamate antiporter-dependent manner.

doi: 10.3892/etm.2021.10710

Figure Lengend Snippet: Figure 4. GPx4 and xCT are required for the fluvastatin‑mediated attenuation of ferroptosis in human endothelial cells. (A) Immunoblots of TNF‑α, IL‑α, 4‑HNE, GPx4 and xCT in the normal, ox‑LDL, ox‑LDL + 10 µM fluvastatin, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + 10 µM fluvastatin, si‑xCT + ox‑LDL and si‑xCT + ox‑LDL + 10 µM fluvastatin treatment groups. (B) Analysis of cell viability using the MTT assay in HUVECs after ox‑LDL, ox‑LDL + 10 µM fluv astatin, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + 10 µM fluvastatin, si‑xCT + ox‑LDL or si‑xCT + ox‑LDL + 10 µM fluvastatin treatments. Columns, means of three experiments; bars, SD. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (C) Histogram of relative TNF‑α/β‑actin levels. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (D) Histogram of relative IL‑α/β‑actin levels. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (E) Histogram of relative 4‑HNE/β‑actin levels. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (F) Histogram of relative GPx4/β‑actin levels. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (G) Histogram of relative xCT/β‑actin levels. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. (H) Representative Transwell migration assay images of HUVECs treated with ox‑LDL, ox‑LDL + 10 µM fluvastatin, si‑GPx4 + ox‑LDL, si‑GPx4 + ox‑LDL + 10 µM fluvastatin, si‑xCT + ox‑LDL and si‑xCT + ox‑LDL + 10 µM fluvastatin. *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL (magnification, 100x). (I) Representative tube formation assay images of HUVECs in the presence of PBS or with 100 µg/ml ox‑LDL, 100 µg/ml ox‑LDL + 10 µM fluvastatin, si‑GPx4 + 100 µg/ml ox‑LDL, si‑GPx4 + 100 µg/ml ox‑LDL + 10 µM fluvastatin, si‑xCT + 100 µg/ml ox‑LDL or si‑xCT + 100 µg/ml ox‑LDL + 10 µM fluvastatin (magnification, 100x). *P<0.05 vs. control group, &P<0.05 vs. si‑GPx4 + ox‑LDL, +P<0.05 vs. si‑xCT + ox‑LDL. ox‑LDL, oxidized low‑density lipoprotein; GPx4, glutathione peroxidase 4; xCT, cystine‑glutamate antiporter; 4‑HNE, 4‑hydroxynonenal; HUVEC, human umbilical vein cell.

Article Snippet: The following primary antibodies were used: 4‐hydroxynonenal (4‐HNE; 1:2,000; ab46544; Abcam), TNF‐α (1:500; WL01581; Wanleibio; Co., Ltd.), IL‐1α (1:2,000; ab134908; Abcam), GPx4 (1:1,000; 14432‐1‐AP; ProteinTech Group, Inc.), xCT (1:1,000; 26864‐1‐AP; ProteinTech Group, Inc.); Caspase‐3/cleaved‐caspase3 (1:500; WL02117; Wanleibio; Co., Ltd.), Bax (1:1,000; WL01637; Wanleibio; Co., Ltd.), Bcl‐2 (1:1,000; WL01556; Wanleibio; Co., Ltd.), β‐actin (1:2,000; WL01845 Wanleibio; Co., Ltd.).

Techniques: Western Blot, MTT Assay, Control, Transwell Migration Assay, Tube Formation Assay

Journal: PeerJ

Article Title: 3,4-dihydroxyphenylethyl alcohol glycoside reduces acetaminophen-induced acute liver failure in mice by inhibiting hepatocyte ferroptosis and pyroptosis

doi: 10.7717/peerj.13082

Figure Lengend Snippet: MDA levels of DAG are decreased compared with APAP. (** p < 0.0001 APAP compared with control, # p = 0.0012 compared with DAG). Proteins extracted from the liver were identified using western blot. DAG decreased the levels of p-ERK and HO-1, whereas it increased the levels of GPX4 (B).

Article Snippet: At room temperature, the membrane was sealed with 5% (w/v) skimmed milk for 2 h. Next, the membrane was incubated with the primary antibody overnight at 4 °C: ERK, p-ERK, HO-1, GPX4, NLRP3, Gasdermin-D (Cell Signaling Technology, MA, USA), Caspase 1 (p20) (Santa Cruz, CA, USA), which would combine HRP.

Techniques: Control, Western Blot

Journal: PeerJ

Article Title: 3,4-dihydroxyphenylethyl alcohol glycoside reduces acetaminophen-induced acute liver failure in mice by inhibiting hepatocyte ferroptosis and pyroptosis

doi: 10.7717/peerj.13082

Figure Lengend Snippet: (A) GSH content in AML12 cells. (**** p < 0.0001 control compared with APAP and APAP compared with adding 150 µM DAG. ** p < 0.05 APAP compared with adding 100 µM DAG) ( n = 3 per group df = 2). (B) ROS in AML12 cells. (C) The expression of IL-1β, IL-18 and NLRP3 in AML12 cells. # p < 0.05 APAP compared with control, ** p < 0.005 APAP compared with adding 100 µM DAG, ** p < 0.005 APAP compared with adding 150 µM DAG (IL-1β). # p < 0.005 APAP compared with control, ** p < 0.05 APAP compared with adding 50 µM DAG, **** p < 0.0001 APAP compared with adding 100 µM DAG, **** p < 0.0001 APAP compared with adding 150 µM DAG (IL-18). # p < 0.005 APAP compared with control, ** p < 0.005 APAP compared with adding 100 µM DAG, ** p < 0.005 APAP compared with adding 150 µM DAG (NLRP3) ( n = 3 per group df = 2). (D) Proteins extracted from AML12 cells were identified using western blot. DAG decreased the levels of p-ERK, HO-1, NLRP3, GSDMD and Caspase1 (p20), while at the same time it increased the levels of GPX4.

Article Snippet: At room temperature, the membrane was sealed with 5% (w/v) skimmed milk for 2 h. Next, the membrane was incubated with the primary antibody overnight at 4 °C: ERK, p-ERK, HO-1, GPX4, NLRP3, Gasdermin-D (Cell Signaling Technology, MA, USA), Caspase 1 (p20) (Santa Cruz, CA, USA), which would combine HRP.

Techniques: Control, Expressing, Western Blot

Journal: Oxidative medicine and cellular longevity

Article Title: Melatonin Inhibits the Ferroptosis Pathway in Rat Bone Marrow Mesenchymal Stem Cells by Activating the PI3K/AKT/mTOR Signaling Axis to Attenuate Steroid-Induced Osteoporosis.

doi: 10.1155/2022/8223737

Figure Lengend Snippet: Figure 1: DEX activates the ferroptotic pathway of BMSCs. (a) ROS staining was performed to test the correlation between different concentrations of DEX and the level of oxidative stress. (b) Quantitative analysis of the number of ROS-positive cells per field in (a). (c) Annexin V-mCherry/SYTOX Green detection kit was used to detect cell death. (d) Quantitative analysis of the percentage of SYTOX green-positive cells in (c). (e–i) BMSCs were treated with various concentrations of DEX for 24 h, and the expressions of system xc-, ACSL4, GPX4, and FSP1were analyzed by western blot and qRT-PCR. (j) Images of immunofluorescence staining of GPX4 in BMSCs after treatment with DEX (10-3 M) for 72 h. (k) Quantification of the fluorescence intensity of GPX4 immunofluorescence positively stained cells. These studies were performed at least 3 biological replicates. Data represent mean ± SD (n = 3). ∗P < 0:05, ∗∗P < 0:01, ∗∗∗P < 0:005 compared with control group.

Article Snippet: To more fully locate and quantitatively examine antigenic substances in tissues, sections were first stained with primary antibodies against GPX4 (1 : 200, orb340797, Biorbyt), CD90 (1 : 500,RT1615, 0 20 40 60 80 G PX 4 po sit iv e c el l nu m be r ( m m –2 ) ⁎⁎⁎ Co nt ro l M od el (i) 0 20 40 60 FS P1 p os iti ve ce ll nu m be r ( m m –2 ) ⁎⁎⁎ Co nt ro l M od el (j) 0 20 40 60 M T1 p os iti ve ce ll nu m be r ( m m –2 ) ⁎⁎⁎ Co nt ro l M od el (k) Figure 2: DEX activates the ferroptotic pathway in SIOP. (a) Quantification of the protein level of MT by ELISA. (b) Images of micro-CT. (c) BMD(g/cm3). (d) BV/TV (%). (e) Images of immunofluorescence double staining of CD90 and GPX4 in bone tissues. (f) Quantitative analysis of the area of GPX4/CD90-positive stains. (g) IHC staining of system xc-, GPX4, FSP1, and MT1, and the IHC-positive cells were marked with black arrows. (h–k) Quantitative analysis of the number of the IHC-positive cells in (g).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Control

Journal: Poultry Science

Article Title: New insights into the spleen injury by mitochondrial dysfunction of chicken under polystyrene microplastics stress

doi: 10.1016/j.psj.2024.103674

Figure Lengend Snippet: The primers involved in this study.

Article Snippet: GPX4 , 1:500 , Boster Biotechnology, China , A02059-1.

Techniques:

Journal: Poultry Science

Article Title: New insights into the spleen injury by mitochondrial dysfunction of chicken under polystyrene microplastics stress

doi: 10.1016/j.psj.2024.103674

Figure Lengend Snippet: The antibodies used in this study.

Article Snippet: GPX4 , 1:500 , Boster Biotechnology, China , A02059-1.

Techniques:

Journal: Poultry Science

Article Title: New insights into the spleen injury by mitochondrial dysfunction of chicken under polystyrene microplastics stress

doi: 10.1016/j.psj.2024.103674

Figure Lengend Snippet: MPs induced ferroptosis in spleen. (A) Western Blot results in ferroptosis-related proteins. (B) Quantitative analysis of GPX4, FTH1, and SLC7A11 protein expression. (C) Ferroptosis-related factors of mRNA expression changes. Data were expressed as mean ± SD, n = 3. *, **, ***, denotes: p < 0.05, 0.01, and 0.001, respectively.

Article Snippet: GPX4 , 1:500 , Boster Biotechnology, China , A02059-1.

Techniques: Western Blot, Expressing

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD surgical tissues show key markers of ferroptosis. ( A ) Heatmap with hierarchical clustering for 211 genes from the FerrDB database that includes known driver, suppressor, and marker ferroptosis genes that were expressed in the RNA-Seq datasets. For each plot, “pearson” was used for the clustering distance and “complete” for the hierarchical clustering method. The location where the representative dataset was collected (Mayo, Russia, or UTSW) and mutation type (Control, no TCF4 repeats [No_Rep] or TCF repeats [TCF4_Rep]) are shown for each sample. ( B ) FSP1 mRNA and protein expression in FECD and control tissues. (C) FTH mRNA expression in control and FECD tissues. (D) GPX4 mRNA expression in FECD and control human tissues. (E) Ferroportin ( FPN1 ) mRNA and protein expression in FECD and control tissues. ( F ) FTL mRNA expression in control and FECD tissues. ( G ) TFR1 mRNA and protein expression in control and FECD surgical tissues. ( H ) Representative immunohistochemistry images of TFR1 localization in non-FECD and FECD donor cornea tissues. (I) 4-HNE protein expression in human surgical samples from patients with FECD (n = 8). All data of mRNA and protein expression are shown as mean ± SEM for n = 12 (Control tissues, 8 pools of 3, each pool contained 3 tissues) and n = 24 (FECD tissues, 8 pools of 3, each pool contained 3 tissues). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Relative gene expression is normalized by β-actin . (J) Cytosolic Fe 2+ in primary CECs isolated from healthy human donor corneas (n = 11, each cornea divided into 2 sections) and FECD surgical explants (n = 7). Data are shown as mean ± SEM; ∗∗p < 0.01, Student's t-test.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Marker, RNA Sequencing Assay, Mutagenesis, Control, Expressing, Immunohistochemistry, Two Tailed Test, Isolation

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD primary and immortalized cell cultures show key marker s of ferroptosis. (A) TFR1 mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. ( B ) FSP1 mRNA expression in primary cells. ( C ) GPX4 mRNA expression in human expanded TCF4 repeat expansion primary cells. (D) FTH mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. (E) FTL mRNA expression in non-FECD and FECD donor primary cells. (F) Representation of median of the fluorescence of DHE showing significant difference in ROS between indicated cells. DHE (FL2 fluorescence) peak of F35T cells shifts to right when compared to B4G12 cells. (G) Representative confocal images showing fluorescence of DHE indicating ROS in the indicated cell lines. (H) Mitochondrial ROS quantified by MitoROS 580 dye in the indicated cells. Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, one-way ANOVA, followed by Tukey's post-hoc test. AMA indicates antimycin-A . (I) GPX4 mRNA and protein expression in HCEC-B4G12 and F35T cells. ( J ) Basal level of lipid peroxidation in HCEC-B4G12 and F35T cells quantified by C11-BODIPY fluorescent probe using flow cytometry. Comparisons of median fluorescence of C11-BODIPY detected in HCEC-B4G12 and F35T cells (10,000 cells). Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, Student's t-test. C11-BODIPY (FL1 fluorescence) peak of F35T cells shifts to right when compares to B4G12 cells. ( K ) Representative confocal images showing fluorescence of reduced and oxidized dye in the indicated cell lines. ( L ) 4-HNE protein expression in HCEC-B4G12 and F35T cells. All data of mRNA and protein expression are shown as mean ± SEM for n = 5–9 (B4G12), n = 5–7 (F35T) and n = 4 (both non-FECD and FECD donor primary cells). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗∗∗p < 0.001. Relative gene expression is normalized by β -actin.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Marker, Expressing, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: Summary of the molecular mechanism of ferroptosis in FECD. Iron enters the cell in ferric form via TFR1-mediated endocytosis. Ferritin stores the excess iron in ferric form, which is nontoxic. The ferric form of iron gets converted to the ferrous form in endosomes. When labile, ferrous iron gets released into the cytosol, it causes lipid peroxidation via Fenton chemistry. UVA irradiation can cause iron release from ferritin which increases the labile iron pool in the cytosol, as well as increases iron-mediated lipid peroxidation, a process known as ferroptosis. GPX4 is the key regulator of ferroptosis, preventing occurrence through scavenging lipid peroxides and reactive oxygen species (ROS). In this study, RSL3 was used to block GPX4 to induce ferroptosis. Ubiquinol, the reduced and active form of coenzyme Q10, is a potent ferroptosis inhibitor that works by scavenging ROS and modulating iron metabolism. Ubiquinol is an essential participant in the FSP1-CoQ10-NAD(P)H pathway, an independent system working in parallel with GPX4 and glutathione to suppress lipid peroxidation and ferroptosis by supporting FSP1 function. Other molecules like DFO and artesunate can prevent ferroptosis by quenching labile toxic ferrous iron, however, are not solely as effective as ubiquinol in preventing ferroptosis.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Irradiation, Blocking Assay

Journal: Environmental Health and Preventive Medicine

Article Title: Chronic noise exposure induces Alzheimer’s disease-like neuropathology and cognitive impairment via ferroptosis in rat hippocampus

doi: 10.1265/ehpm.24-00126

Figure Lengend Snippet: Noise exposure induced ferroptosis in hippocampus of rats. (A–B) Changes in expression levels of GPX4, FTH1, and SLC7A11 in the rat hippocampus (n = 3). (C) Immunohistochemistry of GPX4 in rat hippocampus. (D–G) Changes in levels of MDA, SOD, GSH, and GSH-Px in the rat hippocampus. *, P < 0.05, **, P < 0.01.

Article Snippet: Paraffin sections of rat brain were deparaffinized with primary antibodies mouse GFAP (sc-166458, 1:500; Santa Crutz Biotechnology), rabbit Iba-1 (GB153502-100, 1:500; Servicebio) and rabbit GPX4 (TA376761, 1:100; ORIGENE) were incubated overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry