Journal: Journal of Translational Medicine

Article Title: RBX1 mitigates ferroptosis by inhibiting NCOA4-mediated ferritinophagy and contributes to the attenuation of intervertebral disc degeneration

doi: 10.1186/s12967-025-06412-7

Figure Lengend Snippet: Ferroptosis progressively increased in human NP tissues as IVDD advanced. A Representative MRI images of human NP tissues with different degrees of degeneration. Four discs samples with mild degeneration and four with severe degeneration from eight patients were collected. B H/E and Alcian blue staining of human NP samples in NP tissues from Pfirrmann grades II and V. Scale bar: 25 μm. C , D Representative western blot images of ferroptosis-associated proteins GPX4, FTH1, and SLC7A11, and ECM degradation-associated proteins MMP3 and ADAMTS5 in NP tissues from Mild- and Severe- IVDD groups. The relative band densities were quantified. E , F IHC staining of GPX4 in human nucleus pulposus sections. Scale bar: 25 μm. Quantitative analysis was shown as percentage of GPX4 + cell in NP tissues. G The relative mRNA levels of GPX4 in NP tissues from Mild- and Severe-IVDD groups. H Relative GSH levels in NP tissues from Mild- and Severe-IVDD groups. I Quantification of MDA contents in NP tissues from Mild- and Severe-IVDD groups. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h and then incubated with the following primary antibodies overnight at 4 °C: NCOA4 (HUABIO, ER62707; 1:1000), RBX1 (HUABIO, ER1802-9; 1:500), MDM2 (HUABIO, ER1902-14; 1:1000), FTH1 (HUABIO, ET1610-78; 1:500), GPX4 (HUABIO, ET1706-45; 1:1000), SLC7A11 (Immunoway, YT8130; 1:1000), MMP3 (HUABIO, ER1706-77; 1:500), ADAMTS5 (Immunoway, YN2121; 1:1000), and GAPDH (HUABIO, ET1601-4; 1:10,000).

Techniques: Staining, Western Blot, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: RBX1 mitigates ferroptosis by inhibiting NCOA4-mediated ferritinophagy and contributes to the attenuation of intervertebral disc degeneration

doi: 10.1186/s12967-025-06412-7

Figure Lengend Snippet: Acidic condition induced ferroptosis-associated cell viability decline, ROS accumulation, and ECM degradation in human NPSCs. A Cell viability was detected by an CCK-8 assay in different groups. B , C Representative western blot images of ECM degradation-associated proteins MMP3 and ADAMTS5 in the acidic-treated NPSCs. The relative band densities were quantified. Western blot analysis showing the expression levels of MMP3 and ADAMTS5 as the pH levels decreased from 7.1 to 6.2. D , E Representative western blot images of ferroptosis-associated proteins GPX4 and SLC7A11 in the acidic-treated NPSCs. The relative band densities were quantified. Western blot analysis showing the expression levels of ferroptosis-associated proteins GPX4 and SLC7A11 as the pH levels decreased from 7.1 to 6.2. F , G After treatment with acidic conditions and acidic conditions + Fer-1 for 24 h, NPSCs were labeled with anti-GPX4 antibodies, and representative fluorescence images are shown. Nuclei were stained with DAPI. Scale bar: 50 μm. Quantitative analysis of GPX4 was shown as relative fluoresence intensity. H – L Representative western blot images of ferroptosis-associated proteins GPX4 and SLC7A11, and ECM degradation-associated proteins MMP3 and ADAMTS5 after NPSCs treated with acidic conditions and acidic conditions + Fer-1 for 24 h. The relative band densities were quantified. M , N Representative fluorescence microscopy photomicrographs of intracellular ROS in NPSCs. Scale bar: 50 μm. Quantitative analysis of intracellular ROS was shown as relative fluoresence intensity. O , P JC-1 staining for mitochondrial membrane potential in NPSCs. Scale bar: 200 μm. Quantitative analysis of mitochondrial membrane potential was shown as the ratio of monomer/aggregate. Q Quantification of MDA contents in NPSCs. R Total iron levels in NPSCs. A P -value less than 0.05 was deemed to be statistically significant. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant)

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h and then incubated with the following primary antibodies overnight at 4 °C: NCOA4 (HUABIO, ER62707; 1:1000), RBX1 (HUABIO, ER1802-9; 1:500), MDM2 (HUABIO, ER1902-14; 1:1000), FTH1 (HUABIO, ET1610-78; 1:500), GPX4 (HUABIO, ET1706-45; 1:1000), SLC7A11 (Immunoway, YT8130; 1:1000), MMP3 (HUABIO, ER1706-77; 1:500), ADAMTS5 (Immunoway, YN2121; 1:1000), and GAPDH (HUABIO, ET1601-4; 1:10,000).

Techniques: CCK-8 Assay, Western Blot, Expressing, Labeling, Fluorescence, Staining, Microscopy, Membrane

Journal: Journal of Translational Medicine

Article Title: RBX1 mitigates ferroptosis by inhibiting NCOA4-mediated ferritinophagy and contributes to the attenuation of intervertebral disc degeneration

doi: 10.1186/s12967-025-06412-7

Figure Lengend Snippet: NCOA4-mediated ferritinophay participated in the acidic-induced ferroptosis in human NPSCs. A , B Representative western blot images of TFR, DMT1, FPN1, and NCOA4 in the acidic-treated NPSCs. The relative band densities of NCOA4 were quantified. C The target prediction of NCOA4 using STRING database. D , E Representative western blot images of FTH1 in the acidic-treated NPSCs. The relative band densities were quantified. F Co-IP analysis of the binding between NCOA4 and FTH1. G Representative western blot images of FTH1, GPX4, SLC7A11, MMP3, and ADAMTS5 in NPSCs treated with 3-MA and CQ under acidic condition. H Western blot analysis showing the expression levels of FTH1, GPX4, SLC7A11, MMP3, and ADAMTS5 after NPSCs treated with NCOA4 -siRNA and Control-siRNA under acidic condition. I The expression of NCOA4 and FTH1 and their colocalization in NPSCs were determined by immunofluorescence. Nuclei were stained with DAPI. Scale bar: 50 μm. J NPSCs were labeled with anti-GPX4 antibodies, and representative fluorescence images are shown. Nuclei were stained with DAPI. Scale bar: 50 μm. K Quantification of MDA contents in NPSCs. L Total iron levels in NPSCs. A P -value less than 0.05 was deemed to be statistically significant. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant)

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h and then incubated with the following primary antibodies overnight at 4 °C: NCOA4 (HUABIO, ER62707; 1:1000), RBX1 (HUABIO, ER1802-9; 1:500), MDM2 (HUABIO, ER1902-14; 1:1000), FTH1 (HUABIO, ET1610-78; 1:500), GPX4 (HUABIO, ET1706-45; 1:1000), SLC7A11 (Immunoway, YT8130; 1:1000), MMP3 (HUABIO, ER1706-77; 1:500), ADAMTS5 (Immunoway, YN2121; 1:1000), and GAPDH (HUABIO, ET1601-4; 1:10,000).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Control, Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Journal of Translational Medicine

Article Title: RBX1 mitigates ferroptosis by inhibiting NCOA4-mediated ferritinophagy and contributes to the attenuation of intervertebral disc degeneration

doi: 10.1186/s12967-025-06412-7

Figure Lengend Snippet: Inhibition of RBX1 promotes ferroptosis through NCOA4-mediated ferritinophagy in human NPSCs. A – E Representative western blot images of NCOA4, FTH1, GPX4, and SLC7A11 in NPSCs treated with RBX1 -siRNA and Control-siRNA under acidic condition for 24 h. The relative band densities were quantified. F , G JC-1 staining for mitochondrial membrane potential in NPSCs. Scale bar: 200 μm. Quantitative analysis of mitochondrial membrane potential was shown as the ratio of monomer/aggregate. H , K Representative western blot images of FTH1, GPX4, and SLC7A11 in NPSCs treated with RBX1 -siRNA, NCOA4 -siRNA and Control-siRNA under acidic condition for 24 h. The relative band densities were quantified. L Quantification of MDA contents in NPSCs. M Total iron levels in NPSCs. A P -value less than 0.05 was deemed to be statistically significant. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h and then incubated with the following primary antibodies overnight at 4 °C: NCOA4 (HUABIO, ER62707; 1:1000), RBX1 (HUABIO, ER1802-9; 1:500), MDM2 (HUABIO, ER1902-14; 1:1000), FTH1 (HUABIO, ET1610-78; 1:500), GPX4 (HUABIO, ET1706-45; 1:1000), SLC7A11 (Immunoway, YT8130; 1:1000), MMP3 (HUABIO, ER1706-77; 1:500), ADAMTS5 (Immunoway, YN2121; 1:1000), and GAPDH (HUABIO, ET1601-4; 1:10,000).

Techniques: Inhibition, Western Blot, Control, Staining, Membrane

Journal: Nutrients

Article Title: Hydroxysafflor Yellow A Alleviates Acute Myocardial Ischemia/Reperfusion Injury in Mice by Inhibiting Ferroptosis via the Activation of the HIF-1α/SLC7A11/GPX4 Signaling Pathway

doi: 10.3390/nu15153411

Figure Lengend Snippet: Hydroxysafflor yellow A alleviated ferroptosis by activating the HIF-1α/SLC7A11/GPX4 signaling pathway. ( A ) The levels of ferroptosis-related proteins were assessed by Western blotting ( n = 3). ( B ) The total iron content in cardiac tissue was determined using a kit ( n = 6). ## p < 0.01 vs. the sham group; * p < 0.05, ** p < 0.01 vs. the MI/RI group.

Article Snippet: Anti-HIF-1α antibody (340462), anti-SLC7A11 antibody (382036), and goat antirabbit IgG secondary antibody (511203) were obtained from Chengdu Zen BioScience Co., Ltd. (Chengdu, China).

Techniques: Western Blot