Journal: Molecular Medicine Reports

Article Title: Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor

doi: 10.3892/mmr.2020.11679

Figure Lengend Snippet: AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Article Snippet: Primary antibodies were specific for AhR (1:200; cat. no. sc-133088; Santa Cruz Biotechnology, Inc.), cytochrome P450 family 1 subfamily A member 1 (CYP1A1; 1:500; cat. no. sc-25304; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), superoxide dismutase 3 (SOD-3; 1:100; cat. no. sc-271170; Santa Cruz Biotechnology, Inc.), cystine-glutamate antiporter (xCT, also known as SLC7A11; 1:1,000; cat. no. ANT-111; Alomone Labs), HIF-1α (1:500; cat. no. sc-10790; Santa Cruz Biotechnology, Inc.), LDH-A (1:1,000; cat. no. 2012; Cell Signaling Technology, Inc.), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Activity Assay, Cell Culture, Western Blot, Expressing

Journal: bioRxiv

Article Title: Oxymatrine Induces Ferroptosis in MKN28 Gastric Cancer Cells

doi: 10.1101/2025.11.22.689973

Figure Lengend Snippet: Effect of oxymatrine on the expression of ferroptosis-related proteins in MKN28 cells. Western blot analysis of ACSL4, GPX4, and SLC7A11 protein expressions in each group.

Article Snippet: Oxymatrine (Shanghai Aladdin Biochemical Technology Co., Ltd.); RPMI 1640 cell culture medium (Gibco); penicillin-streptomycin double antibody (Gibco); CCK-8 kit (Shanghai Yeasen Biotechnology Co., Ltd.); iron ion detection kit (Nanjing Jiancheng Bioengineering Institute); protein extraction kit (Beyotime Biotechnology Co., Ltd.); GPX4, SLC7A11, and Nrf2 antibodies were purchased from Proteintech Group, Inc.

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: Primers for qRT-PCR.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Reverse Transcription

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: GW4869 attenuates the degradation of ferroptosis-associated proteins and the secretion of exosomes in atrial tissue of rapid atrial pacing in canines. (a) Representative gel bands depicting exosome markers and ferroptosis-associated proteins expression using specific antibodies. GAPDH was used as the loading control. (b) Levels of CD63, CD81, and TSG101. ( n =5). (c) Levels of FTH1, GPX4, and SCL7A11. ( n =5). (d, f) Representative images of inflammatory cell infiltration, ferroptosis, fibrosis, and SLC7A11 as reflected by H&E staining, Prussian staining, Masson staining, and immunohistochemistry. ( n =3). (e, g) Representative images of immunofluorescence staining for FTH1 protein in canines atrial tissue stimulated by rapid pacing with or without GW4869. ( n =3). (h) RT-PCR analysis of FTH1, GPX4, and SLC7A11 expression normalized with GAPDH. ( n =5). Data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. Sham group; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. Pacing group.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Expressing, Control, Staining, Immunohistochemistry, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: Chronological changes in ferroptosis-associated genes and oxidative stress levels in h9c2 cells after rapid pacing. (a–d) Chronological changes of FTH1, GPX4, SLC7A11, and FTL miRNAs after rapid pacing. (e, f) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (g, h) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells stimulated by rapid pacing for 72 h and treated with or without Fer-1. (i, k) Representative images of immunofluorescence staining for FTH1 proteins in h9c2 cells stimulated by rapid pacing for 48 h with or without Fer-1. (j, l) Representative images of reactive oxygen species by fluoroscopy stimulated by rapid pacing for 48 h with or without Fer-1. (m, n) Mitochondrial membrane potential was detected through flow cytometry in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (o) Total iron level in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (p) MDA level in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (q, r) Representative gel bands depicting ion channel expression in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. Data are presented as the mean ± SD, n =3. Statistical significance was determined using Student's t test (a–d) or one-way ANOVA with a post hoc Dunnett test (e–r). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. Pacing group.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Flow Cytometry, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: Rapid pacing primary cardiac fibroblast-derived exosomes aggravate the ferroptosis of h9c2 cells. (a) Morphology of primary cardiac fibroblast-derived exosomes (CF-exos) using transmission electron microscopy. (b) Mean exosomes diameter shown by ZetaView System. (c) Exosomes were isolated from culture supernatant from CFs, dyed with phalloidin (green) and cocultured with h9c2 cells, then dyed with PKH26 (red) and viewed with fluoroscopy. (d, e) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells with or without Fer-1 incubated with CF-exos for 48 h. CFs treated with or without GW4869 and stimulated by rapid pacing for 48 h and its exosomes were isolated from culture supernatant. (f) Total iron level in h9c2 cells incubated with CF-exos. (g) MDA level in h9c2 cells incubated with CF-exos. (h, i) Mitochondrial membrane potential was detected through flow cytometry in h9c2 cells incubated with CF-exos. (j, k) Reactive oxygen species was detected through flow cytometry in h9c2 cells incubated with CF-exos. Data are presented as the mean ± SD, n =3. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. pacing-CF-exos group. Abbreviations: control-CF-exos: h9c2 cells incubated with control-CF-exos; pacing-CF-exos: h9c2 cells incubated with CF-exos stimulated by rapid pacing 48 h; pacing-CF-exos+Fer-1: h9c2 cells treated with Fer-1 (20 μ M) and incubated with CF-exos stimulated by rapid pacing 48 h; GW4869-pacing-CF-exos: h9c2 cells incubated with CF-exos treated with GW4869 (20 μ M) and stimulated by rapid pacing 48 h.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Expressing, Incubation, Membrane, Flow Cytometry, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: miR-23a-3p accelerates ferroptosis in h9c2 cells via SLC7A11 downregulation. (a) RT-PCR analysis of miR-23 in h9c2 cells transfected with miR-23a-3p inhibitor and miR-23-3p mimics, respectively. (b) RT-PCR analysis of SLC7A11 expression normalized with GAPDH after transfected with mimics and inhibitor-miR-23a-3p, respectively. (c) Total iron level in h9c2 cells transfected with mimics-miR-23a-3p. (d) MDA level in h9c2 cells transfected with mimics-miR-23a-3p. (e, f) Mitochondrial membrane potential was detected through flow cytometry in h9c2 cells transfected with mimics-miR-23a-3p and treated with or without Fer-1. (g, h) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells transfected with mimics-miR-23a-3p and treated with or without Fer-1. Data are presented as the mean ± SD, n =3. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. ∗, ∗∗, and ∗∗∗ indicate P <0.05, 0.01, and 0.001, respectively. Abbreviations: mimics: h9c2 cells transfected with mimics-miR-23a-3p (25 μ M) for 48 h; inhibitor: h9c2 cells transfected with inhibitor-miR-23a-3p (30 μ M) for 48 h.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Membrane, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: Inhibitor-miR-23a-3p protects h9c2 cells from ferroptosis by upregulating SLC7A11. (a, b) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells transfected with inhibitor-miR-23a-3p and stimulated by rapid pacing 48 h. ( n =3). (c) Total iron level in h9c2 cells transfected with inhibitor-miR-23a-3p and stimulated by rapid pacing 48 h. ( n =3). (d) MDA level in h9c2 cells transfected with inhibitor-miR-23a-3p and stimulated by rapid pacing 48 h. ( n =3). (e, f) Early cell apoptosis was detected through flow cytometry in h9c2 cells transfected with inhibitor-miR-23a-3p and stimulated by rapid pacing 48 h. ( n =3). (g, h) Representative images of reactive oxygen species by fluoroscopy in h9c2 cells transfected with inhibitor-miR-23a-3p and stimulated by rapid pacing 48 h. ( n =3). (i) Target sequence of miR-23a-3p in the wild-type (WT) SLC7A11 3′ UTR and sequence of mutated (Mut) SLC7A11 3′ UTR predicted by starBase v2.0. (j) Measurement of firefly luciferase activity normalized to Renilla luciferase activity in 293 T cells. ( n =5). Data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. ∗, ∗∗, and ∗∗∗ indicate P <0.05, 0.01, and 0.001, respectively. Abbreviations: inhibitor: h9c2 cells transfected with inhibitor-miR-23a-3p (30 μ M); inhibitor+pacing: h9c2 cells transfected with inhibitor-miR-23a-3p and pacing for 48 h (30 μ M).

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Expressing, Transfection, Flow Cytometry, Sequencing, Luciferase, Activity Assay

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 on System X c − gene expression in U373 cells. Cells were treated with Aβ 25-35 (50 µM) for 4, 8, and 16 h. After incubation at 37 °C, cells were homogenized, and total RNA has been purified to evaluate mRNA content of System X c − by RT-qPCR. Results are computed relative to GAPDH content, taken as a housekeeping gene. Bars are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. * p ≤ 0.05 vs. CTRL.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Expressing, Incubation, Purification, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 on System X c − protein expression in U373 cells. Cells were treated with Aβ 25-35 (50 µM) for 24 h. The graph shows the densitometric analysis of the western blots for each sample. Data are computed relative to the internal housekeeping gene (actin) and are the means ± SEM from three separate experiments, each carried out in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. ** p ≤ 0.01 vs. CTRL.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Expressing, Western Blot

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: System X c − localization in Aβ 25-35 -treated U373. Cells were treated with Aβ 25-35 (50 µM) for 24 h and subjected to immunofluorescence staining using anti-System X c − 1:100 (OriGene, green) antibodies as reported in Materials and Methods. Nuclei (blue) are stained with Hoechst 33342.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Immunofluorescence, Staining

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 and System X c − activity on release of extracellular glutamate in differentiated SH-SY5Y/U373 co-cultures. Cells were treated for 24 h and the glutamate assay was performed as specified in the Materials and Methods. The graph shows the extracellular release of glutamate (µM). Data are calculated relative to a glutamate standard curve and are the means ± SEM from three separate experiments, each carried out in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. *** p ≤ 0.001 between CTRL and Aβ 25-35 ; ** p < 0.01 between Aβ 25-35 and Aβ 25-35 + SSZ.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activity Assay, Glutamate Assay

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Role of System X c − and NMDA receptor activation on the viability of Aβ-treated SH-SY5Y differentiated cells. ( A ) SH-SY5Y cells were grown in mono-cultures and treated for 24 h with Aβ 25-35 (50 μM). ( B ) SH-SY5Y cells were co-cultured with U373 cells and treated for 24 h with Aβ 25-35 (50 μM) alone or in the presence of either SSZ (300 μM) or MK801 (10 μM). MTT cell viability assay was carried out as specified in Materials and Methods. The histograms show the percentage of living cells, and the rate of reduction was calculated by setting the control (CTRL) equal to 100%. Values are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to determine significant differences. NS not significant; ** p ≤ 0.01 between CTRL and Aβ 25-35 ; ** p ≤ 0.01 between Aβ 25-35 and Aβ 25-35 + SSZ; * p ≤ 0.05 between Aβ 25-35 and Aβ 25-35 + MK801.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activation Assay, Cell Culture, Viability Assay

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Proposed model for Aβ-dependent neurodegeneration during AD. In astrocytes, Aβ 25-35 elicits an antioxidant response by transcriptionally inducing Nrf2-driven ARE genes, such as CAT, HO-1, SOD1, SOD2, GPX3, GCLC, and System X c − . While the L-cystine import through System X c − is crucial to protection from oxidative stress (e.g., GSH production), the export of glutamate may cause neurodegeneration through the activation of NMDAr on neuronal cells. For more details see text.Abbreviations: Aβ, amyloid-β; ARE, antioxidant responsive element; CAT, catalase; GCLC, glutamate-cysteine ligase; GPX3, glutathione peroxidase; GSH, reduced glutathione; HO-1, heme-oxygenase-1; NMDAr, N-methyl-D-aspartate receptor; Nrf2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activation Assay