Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Tfh cell RNA-Seq data analysis. Note: ( A ) The volcano plot presents the expression of DEGs based on RNA-Seq sequencing analysis of three Tfh cell samples (CD4 + ICOS + CXCR5 +) from MRL/lpr and MRL/Mpj mice. Blue dots represent downregulated genes, red dots represent upregulated genes, and black dots represent genes without significant differences. B The hierarchical clustering heatmap displays the top 20 DEGs. C The bar chart shows the results of GO functional analysis and KEGG pathway enrichment analysis of DEGs, including biological processes (BP), cellular components (CC), and molecular functions (MF), as well as the associated network diagram. D The Venn diagram illustrates the intersection of DEGs and the marker genes of Tfh cells (Cluster 7). E The box plot represents the expression of HMGA1 in RNA-Seq data. F Western blot analysis was conducted to examine the protein level of HMGA1 in Tfh cells, with each experiment involving 6 mice. Cell experiments were repeated three times, with three replicates each time. *** indicates P < 0.001 compared to MRL/Mpj mice

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: RNA Sequencing, Expressing, Sequencing, Functional Assay, Marker, Western Blot

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Impact of HMGA1 knockdown or overexpression on Tfh cells. Note: ( A ) Western blot analysis confirms the knockdown and overexpression effect of HMGA1. B The CCK8 experiment measures the influence of HMGA1 on the proliferation capability of Tfh cells. C Statistical results of iron content in Tfh cells in various groups using a detection kit. D Measurement of lipid peroxidation levels in Tfh cells using spectrophotometry. E Levels of MDA in each group were detected. F GSH/GSSG ratio in Tfh cells after different treatments. G Electron microscopic images of Tfh cells (Scale bar = 2 μm/50 nm). The scale bar in (G) represents 1 μm. Cell experiments were repeated three times, with three replicates each time. *** indicates a P < 0.001

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: Knockdown, Over Expression, Western Blot, Spectrophotometry

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: RNA-Seq data analysis of Tfh cells with HMGA1 knockdown and control. Note: ( A ) The volcano plot displays the expression of DEGs based on RNA-Seq sequencing of Tfh cell samples, with three samples, each in the HMGA1 knockdown group and control group. Blue dots represent downregulated genes, red dots represent upregulated genes, and black dots represent genes without significant differences. B The hierarchical clustering heatmap shows the top 10 DEGs. C DEGs underwent GO functional analysis and KEGG pathway enrichment analysis at the biological process (BP), cellular component (CC), and molecular function (MF) levels, presented as a bar chart and associated network diagram

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: RNA Sequencing, Knockdown, Control, Expressing, Sequencing, Functional Assay

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Investigation of the molecular mechanism of HMGA1regulation on EZH2. Note: ( A ) Heatmap showing the expression patterns of STAT3 and GPX4 genes in Tfh cells with HMGA1 knockdown compared to control group based on RNA-seq data; ( B ) Effects of HMGA1 knockdown or overexpression on the expression levels of STAT3 and GPX4 detected by PCR; ( C ) Venn diagram demonstrating the intersection analysis between DEGs and upstream transcriptional regulatory factors of STAT3; ( D ) Analysis of EZH2 expression levels based on the transcriptome dataset; ( E ) Western blot analysis of EZH2 protein expression levels in Tfh cells with HMGA1 knockdown or overexpression; ( F ) Schematic representation of HMGA1 binding sites; ( G ) Analysis of HMGA1 binding sites on the EZH2 promoter, with the numbers indicating the start positions of the binding sites, and the upper and lower positions indicating the presence of the binding site on the sense and antisense strands, respectively; ( H ) Assessment of the impact of HMGA1 knockdown or overexpression on the EZH2 promoter activity through a luciferase reporter gene assay. In all the figures, * P < 0.05, ** P < 0.01, and *** P < 0.001, while ns P > 0.05. Cell experiments were repeated three times, with three replicates each time

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: Expressing, Knockdown, Control, RNA Sequencing, Over Expression, Western Blot, Binding Assay, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Investigation of the molecular mechanism of HMGA1 regulation on Tfh cell ferroptosis. Note: ( A ) Western blot analysis to confirm the knockdown and overexpression effects of EZH2; ( B ) Measurement and statistical analysis of iron content in Tfh cells of each group using a reagent kit; ( C ) Detection of lipid peroxidation levels in Tfh cells through spectrophotometric method; ( D ) Measurement of MDA content in different groups; ( E ) Comparison of GSH/GSSG ratio in Tfh cells after different treatments; ( F ) Electron microscopy images of Tfh cells and quantitative analysis of mitochondria within the cells; ( G - H ) Western blot analysis of HMGA1, EZH2, P-STAT3, STAT3, and GPX4 protein levels in Tfh cells of different groups. In all the figures, * P < 0.05, ** P < 0.01, and *** P < 0.001. All cell experiments were repeated three times, with three replicates each time

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: Western Blot, Knockdown, Over Expression, Comparison, Electron Microscopy

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Analysis of the impact of the EZH2/STAT3/GPX4 axis on SLE progression. Note: ( A ) Assessment of anti-dsDNA titers in mouse serum using ELISA; ( B ) Assessment of complement C3 content in serum using ELISA; ( C ) Representative images and length quantification of axillary lymph nodes in mice; ( D ) Representative images and length quantification of spleens in mice; ( E – F ) FCM analysis to measure the quantity of Tfh cells in mouse blood; ( G ) Measurement of lipid peroxidation levels in Tfh cells through spectrophotometric method; ( H ) Detection of MDA content in each group; ( I ) Comparison of GSH/GSSG ratio in Tfh cells after different treatments; ( J - K ) Western blot analysis of HMGA1, EZH2, P-STAT3, STAT3, and GPX4 protein levels in Tfh cells of different groups. * P < 0.05, ** P < 0.01, and *** P < 0.001, while ns P > 0.05 in the figures

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Western Blot

Journal: Cell Biology and Toxicology

Article Title: HMGA1 influence on iron-induced cell death in Tfh cells of SLE patients

doi: 10.1007/s10565-024-09955-5

Figure Lengend Snippet: Schematic representation of the molecular mechanism of HMGA1/EZH2/STAT3/GPX4 axis regulating Tfh cell ferroptosis and its impact on SLE. Image generated using Biorender (BioRender)

Article Snippet: Subsequently, primary antibodies against HMGA1 (dilution 1:50, 3935S, CST, USA) and EZH2 (dilution 1:50, 5246 T, CST, USA) were added, dissolved in PBS, and left to react overnight at 4 °C.

Techniques: Generated