Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: NSUN2 suppressed ferroptosis in LPS-induced SIMI. Cellular A , Fe 2 + level, B , GSH content, and C , relative ROS production in each group were determined by commercial kits; D , Western blot was performed to assess the protein levels of NCOA4, FTH1, and GPX4 in each group Fe 2+ , ferrous iron; GSH, glutathione; ROS, reactive oxygen species; NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Western Blot

Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: NSUN2 inhibited NCOA4 expression in a m 5 C-dependent manner. A , The expression of NCOA4, FTH1, and GPX4 after NSUN2 overexpression in H9c2 cells was detected by RT-qPCR; B , MeRIP-qPCR assay was performed to detect m 5 C levels of NCOA4, FTH1, and GPX4 in vector and NSUN2 groups; C , RIP assay evaluated the interaction of NSUN2 and NCOA4 in H9c2 cells; D , Random Forest method was used to predict possible m 5 C sites of NCOA4; Dual-luciferase gene reporter assay evaluated the binding of NSUN2 and NCOA4 at sites E , 501, F , 1138, and G , 2917; H , RNA stability assay was used to detect the NCOA4 expression when actinomycin D treated at different time points (0, 4, 8, and 12 h) after NSUN2 overexpression NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase; NSUN, NOL1/NOP2/SUN domain; RT-qPCR, reverse transcription-polymerase chain reaction; MeRIP, Methylated RNA immunoprecipitation; RIP, RNA immunoprecipitation; m 5 C, 5-methylcytosine

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Reporter Assay, Binding Assay, Stability Assay, Reverse Transcription, Polymerase Chain Reaction, Methylation, RNA Immunoprecipitation

Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: Overexpressing NCOA4 downregulated cell viability and upregulated LDH activity, inflammation, and ferroptosis in LPS-induced SIMI. A , NCOA4 mRNA level was detected by RT-qPCR; B , The cell viability of H9c2 cells in each group was analyzed by CCK-8 assay; C , Determination of LDH enzyme activity in each group; D , The contents of TNF-α, IL-6, and IL-8 in each group was evaluated by ELISA; Cellular E , Fe 2 + level, F , GSH content, and G , relative ROS production in each group were determined by commercial kits; H , The protein levels of NCOA4, FTH1, and GPX4 in each group was analyzed by Western blot RT-qPCR, reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor-α; IL, interleukin; Fe 2+ , ferrous iron; GSH, glutathione; ROS, reactive oxygen species; NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Activity Assay, Quantitative RT-PCR, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Cell Counting

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Prognostic values of FTH1, FTL, and FTH1/FTL ratio in the MS data set of 126 TNBC tumors. A , Expression of FTH1 is significantly higher in good ( n = 82) prognostic TNBC patients, but there is no significant difference in expression of FTL and FTH1/FTL ratio between the two groups of patients; B , Expression of FTH1 significantly correlates to metastasis-free survival in Kaplan-Meier survival analyses (Q1- Q4 = first, second, third and fourth quartile. HR = hazard ratio, CI = confidence interval).

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: GSEA analyses reveal TNBC prognosis and FTH1-associated gene sets. A , Regulation of mesenchymal (M) subtype specific genes in TNBC patients; B , Regulation of immunomodulatory subtype (IM) specific genes in TNBC patients; C , Overlap between TNBC prognosis-associated pathways and FTH1 expression related pathways.

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Visualization of the network between TNBC prognosis and FTH1 related pathways in EnrichmentMap software. All the enriched pathways were divided into 4 major subnetworks: cytokine signaling, link between innate and adaptive immunity, adaptive immunity, and cell death. Node size represents the gene-set size. Density of node color is related to enrichment of pathways. Thickness of connective lines indicates the degree of overlap between two gene-sets. Top-ranking pathways based on the normalized enrichment score (NES) were listed under each of the subnetworks.

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Software

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Over-represented network from all the proteins correlated with FTH1 expression. A , A hierarchical cluster of proteins significantly correlating with FTH1 expression forms two distinct clusters, representing to those which show positive (orange bar) and negative (green bar) correlation. The red and green squares highlight two highly correlated subclusters; B , The red subcluster positively correlates with FTH1 expression; C , The green subcluster negatively correlates with FTH1 expression; D , The protein interaction map from the red subcluster is associated with biological process of “cell morphogenesis and adhesion”; E , The protein interaction map from the green subcluster is associated with the biological process of “cell cycle and proteasome.”

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Subcellular localization of FTH1 and its prognostic significance. A , Cytoplasmic and nuclear staining of FTH1 on TMA and their corresponding cutoff points for Kaplan-Meier analyses; B , Kaplan-Meier analyses show that >75% of tumor cells positive for cFTH1 was associated with favorable prognosis (left panel), whereas >1% positive nFTH1 was related to unfavorable prognosis of TNBC patients (right panel); C , A significant correlation (Spearman correlation) was observed between total FTH1 as measured by LC-MS and cFTH1 staining (left panel), but not nFTH1 staining.

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Univariate and multivariate analyses of cFTH1 and nFTH1 related to metastasis-free survival (n = 147)

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Expressing, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Ferritin Heavy Chain in Triple Negative Breast Cancer: A Favorable Prognostic Marker that Relates to a Cluster of Differentiation 8 Positive (CD8+) Effector T-cell Response *

doi: 10.1074/mcp.M113.037176

Figure Lengend Snippet: Accumulation of CD8+ T-lymphocytes in tumor regions with expression of cFTH1. A , IHC staining of FTH1, CD3, CD4 and CD8 in TNBC whole tissue sections (yellow arrows: CD4+ T-lymphocytes; red arrows: CD8+ T-lymphocytes); B , Box plots summarize the observation in (A). CD4+/CD8+ ratio was significantly increased in TNBC samples with high expression of cFTH1. Data are presented as the median ± interquartile range (Mann-Whitney U test, n = 15 per group); C , A proposed functional model of cFTH1 in the context of the antigen processing and presentation pathway: Ferritin complex captures intracellular Fe 2+ ions (green dots) and converts them into Fe 3+ ions (yellow dots). Accumulation of Fe 3+ iron in tumor cells increases in response to inflammation, and IFN γ (red dots) can enhance the inflammatory response of tumor cells. Together, inflammatory signals can enhance processing of cytosolic antigens through the proteasome, heat shock proteins (HSP), and antigen peptide transporters (TAP), resulting in antigen presentation by major histocompatibility complex class I (MHC I). This in turn attracts CD8+ T-lymphocytes that recognize tumor cells and induce the apoptotic process. On the other hand, cFTH1 can be transported from the cytoplasm to the nucleus. nFTH1 protects from DNA damage caused by reactive oxygen species (ROS) (dashed arrow). Solid lines: observed interactions; Dashed line: hypothetical events; Green arrows: molecular functions of cFTH1; Red arrow: molecular function of nFTH1; Black arrows: pathways driven by FTH1.

Article Snippet: Tissue sections of 4 μm were incubated for 1 h at room temperature with anti-FTH1 antibody at a dilution of 1:100 against residues near the C terminus of the protein (rabbit-anti-human monoclonal antibody, clone name: EPR3005Y, GeneTex Inc., Irvine, CA, USA).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY, Functional Assay, Immunopeptidomics

Journal: Clinical and Translational Medicine

Article Title: Integrated multi‐omics profiling landscape of organising pneumonia

doi: 10.1002/ctm2.1782

Figure Lengend Snippet: Reprogramming of lipid metabolism in alveolar macrophages promotes ferroptosis. (A‒D) Violin plot showing the expression levels of FTL (B) and FTH1 (C) in the 13 clusters of healthy control (HC) and organising pneumonia (OP) tissues from the single‐cell RNA sequencing (scRNA‐seq) data. (E) Network of differentially expressed proteins between CON and siCD36 groups (red represents upregulation and green represents downregulation). (F) Gene set enrichment analysis (GSEA) analysis revealed that the changed proteins were mainly enriched in lung fibrosis, mitochondria‐related metabolism, fatty acid metabolism, immune system, cell death and ferroptosis‐related pathways by knocking down CD36. (G) Heatmap showing the differentially expressed proteins related to ferroptosis in alveolar macrophages by knocking down CD36. (H and I) Western blot assay was used to detect the expression of CD36, SLC3A2, GPX4, FTH1, FTL, CES1 and SCD1 in alveolar macrophages. (J) Electron micrograph of alveolar macrophages of OP mouse model (magnification, 30 000×; red arrow, mitochondrion; red rectangle, nucleus). (K) Quantification of perinuclear mitochondrial area, number of mitochondria per square micron, mitochondrial circularity index, cristae number, cristae area, cristae volume and cristae score from (J). (L) The concentration of Fe 2+ , malondialdehyde (MDA) and glutathione (GSH) in alveolar macrophages of OP mouse model. (M and N) Electron micrograph of alveolar macrophages with siCD36 treatment. Quantification of perinuclear mitochondrial area, number of mitochondria per square micron, mitochondrial circularity index, cristae number, cristae area, cristae volume and cristae score from (M). (O) The levels of Fe 2+ , MDA and GSH in alveolar macrophages with siCD36 treatment. Statistical significance is indicated by asterisks; *** p ≤ .001.

Article Snippet: The PVDF membrane was treated with rabbit anti‐SCD1 polyclonal antibody (1:1000, 28678‐1‐AP, Proteintech Group), rabbit anti‐CD36 polyclonal antibody (1:1000, 18836‐1‐AP, Proteintech Group), rabbit anti‐CES1 polyclonal antibody (1:1000, 16912‐1‐AP, Proteintech Group), rabbit anti‐β‐actin monoclonal antibody (1:1000, 66009‐1 Ig, Proteintech Group), rabbit anti‐FTH1 monoclonal antibody (1:1000, R382071, Chengdu Zen Bioscience) or mouse anti‐FTL monoclonal antibody (1:1000, 220988, Chengdu Zen Bioscience), mouse anti‐GPX4 monoclonal antibody (1:1000, 67763‐1‐Ig, Proteintech Group), rabbit anti‐CD98/SLC3A2 polyclonal antibody (1:1000, 15193‐1‐AP, Proteintech Group), rabbit anti‐vimentin polyclonal antibody (1:1000, 10366‐1‐AP, Proteintech Group), rabbit anti‐N‐cadherin polyclonal antibody (1:1000, 22018‐1‐AP, Proteintech Group) and rabbit anti‐E‐cadherin polyclonal antibody (1:1000, 20874‐1‐AP, Proteintech Group) followed by overnight incubation at 4°C in a shaker.

Techniques: Expressing, Control, RNA Sequencing Assay, Western Blot, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Evolution of infection by Mycobacterium avium in mice deficient in H-ferritin in myeloid cells. Fth1 +/+ (blue) and Fth1 −/− (red) mice were intravenously infected with 10 6 CFU of M. avium 25291. ( A ) Animals’ body weight throughout the experiment. Data are expressed as percentage of the initial weight, presented as the mean ± SD of 5 to 8 animals per group. Empty circles: noninfected; filled circles: infected. ( B ) Bacterial burden 60 days post-infection in the liver, spleen, and bone marrow of 8 Fth1 +/+ and 7 Fth1 −/− mice. Bars represent the mean, and circles represent each animal. ( C ) Kaplan–Meier survival curve corresponding to 12 Fth1 +/+ and 16 Fth1 −/− animals. Mice were euthanized when they lost 20% of the initial weight (between day 43 and day 152 post infection). ( D ) Bacterial burden in the liver, spleen, and bone marrow, at the time of euthanasia in the survival curve, of 12 Fth1 +/+ and 16 Fth1 −/− mice. Bars represent the mean and circles represent each animal. Statistics: multiple t -test in ( A , B , D ); log-rank (Mantel–Cox) test in ( C ). ** p < 0.01 and *** p < 0.001 when comparing Fth1 −/− with Fth1 +/+ mice.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Infection

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Inflammatory response in mice infected with M. avium . Fth1 +/+ (blue) and Fth1 −/− (red) mice were intravenously infected with 10 6 CFU of M. avium 25291 and euthanized 60 days later. ( A ) TNF-alpha, ( B ) IFN-gamma, and ( C ) IL-6 were measured in the serum, using a cytometric bead array kit. Bars represent the mean, and the circles represent individual values of 4 to 8 animals per group. Statistics: two-way ANOVA followed by Sidak multiple-comparison post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 when comparing Fth1 +/+ to Fth1 −/− mice. ## p < 0.01, ### p < 0.001 when comparing infected to noninfected mice of the same genotype. ( D – G ) Representative images of H&E-stained liver sections from noninfected (NI) and infected (60 dpi) Fth1 +/+ and Fth1 −/− mice. Representative granulomas are outlined. Scale bar: 200 µm.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Infection, Comparison, Staining

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Impact of infection on iron distribution. Sera from Fth1 +/+ (blue) and Fth1 −/− (red) mice infected for 60 days with M. avium 25291 (60 dpi) or from noninfected mice (NI), were used to measure ( A ) iron, ( B ) transferrin saturation, and ( C ) total ferritin ( n = 5 to 8). ( D , E ) Serum FTL and FTH1 subunits were measured by ELISA ( n = 2 to 4). ( F ) Liver sections were collected, and the iron concentration was determined by atomic absorption spectrometry ( n = 5 to 8). Bars represent the mean, and circles represent each animal. Statistics: two-way ANOVA followed by Sidak multiple-comparison post hoc test. ** p < 0.01, *** p < 0.001 when comparing Fth1 −/− with Fth1 +/+ mice in the same experimental condition. # p < 0.05, ## p < 0.01, ### p < 0.001 when comparing infected to noninfected mice of the same genotype. ( G – J ) Perls’ staining of liver sections was used to evaluate iron distribution. Arrows indicate regions of iron accumulation (blue staining). Scale bar = 200 µm.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison, Staining

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Effect of iron overload on the growth of M. avium in the liver. Groups of Fth1 +/+ (blue) and Fth1 −/− (red) mice were intraperitoneally injected with 10 mg of iron, in the form of iron–dextran, 10 days before intravenous infection with 10 6 CFU of M. avium 25291. Sixty days after infection, noninfected (NI) and infected (60 dpi) mice were euthanized. ( A ) Liver iron was quantified by atomic absorption spectrometry ( n = 4 to 8 per group). ( B ) Bacterial burden in the livers, 60 days after infection ( n = 6 to 8 per group). The bars represent the mean of the group, and each circle corresponds to one animal. Statistics: two-way ANOVA followed by Sidak multiple-comparison post hoc test. *** p < 0.001 when comparing Fth1 −/− to Fth1 +/+ mice in the same experimental condition. @ p < 0.001 when comparing iron-overloaded with non-iron-overloaded mice of the same genotype and infection status.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Injection, Infection, Comparison

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Iron-related gene expression induced by mycobacterial infection.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Gene Expression, Infection

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Effects of infection and myeloid Fth1 deficiency in iron and ferroportin distribution in the liver. Liver sections were obtained from ( A , B ) noninfected mice or ( C – H ) mice infected with M. avium for 60 days. Sections were stained for ( A – D , G , H ) ferroportin, using immunohistochemistry (brown staining) or for ( E , F ) iron, using the Perls’ method (blue staining). ( E – H ) Mice were intraperitoneally injected with 10 mg of iron, as iron–dextran, 10 days before infection. Representative images of at least three animals per experimental condition. Representative granulomas are outlined. Scale bar: 200 µm.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Infection, Staining, Immunohistochemistry, Injection

Journal: International Journal of Molecular Sciences

Article Title: H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

doi: 10.3390/ijms23010269

Figure Lengend Snippet: Primers sequences.

Article Snippet: FTH1 and FTL were quantified by ELISA in the mice serum samples, using rabbit polyclonal anti-mouse FTH1 or FTL (DIBIT-IRCCS San Raffaele Scientific Institute, Milan, Italy) as primary antibodies, and biotinylated rabbit polyclonal anti-mouse FTH1 or FTL as the secondary antibodies.

Techniques: Sequencing

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: Diagnostic value of BCAR3 methylation in distinguishing thyroid cancers from benign thyroid nodules. The methylation levels of six CpG sites within the BCAR3 gene were utilized to generate a prediction probability via logistic regression with age and sex adjusted. ( a ) Center I, ( b ) Center II, ( c ) All participants combined center I and II. Abbreviations: AUC, area under the curve. CI, confidence interval.

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Diagnostic Assay, Methylation

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: Comparison of BCAR3 methylation across thyroid tumor subtypes. ( a ) Boxplot visualization of BCAR3 methylation levels across different subtypes. ( b ) Matrix of p values from pairwise subgroup comparisons (Kruskal–Wallis test with Dunn’s post hoc test). Significant differences are indicated by gray shading, where a darker shade represents a smaller p value. The shading key is: white ( p ≥ 0.05), light gray ( p < 0.05), and dark gray ( p < 0.001). Abbreviations: PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; ATC, anaplastic thyroid carcinoma.

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Comparison, Methylation

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: BCAR3 expression in benign and malignant thyroid tissues and three different cell lines (Nthy-ori 3-1, normal thyroid cell line; TPC-1, and KTC-1, thyroid cancer cell lines). ( a , b ) BCAR3 expression levels in BTN (n = 9) and TC (n = 9) tissues detected by immunohistochemistry ( a ). The bar graph quantifies the mean intensity of BCAR3 staining ( b ). Statistical significance was determined by an unpaired t -test. ( c ) BCAR3 methylation levels of 3 different cell lines (n = 3 biological replicates), detected by MALDI-TOF mass spectrometry. Statistical comparisons of each cancer cell line (TPC-1, KTC-1) against the normal thyroid cell line (Nthy-ori 3-1) were performed using unpaired t -tests. ( d ) The relative mRNA levels of BCAR3 normalized to the internal control (GAPDH) detected by quantitative real-time PCR (n = 3). Statistical comparisons of each cancer cell line against the normal thyroid cell line were performed using unpaired t -tests. ( e , f ) BCAR3 protein expression detected by Western blot analysis, with GAPDH as a loading control ( e ). The quantitative assessment of protein expression levels is presented as the relative intensity of each protein band (n = 3). Statistical comparisons of each cancer cell line against the normal thyroid cell line were performed using unpaired t -tests ( f ). Data in all bar graphs are presented as mean with SD. * p value < 0.05; ** p value < 0.01; **** p value < 0.001. Abbreviations: TC, thyroid cancer; BTN, benign thyroid nodule. The uncropped blots are shown in .

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Staining, Methylation, Mass Spectrometry, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: RNA interference target screening and BCAR3 knockdown effect in TPC-1 and KTC-1 cells. The abbreviations of the four groups in different cells are as follows: WT, wild type; NC, negative control with si-NC treated; KD, knockdown by si- BCAR3 ; AC, knocked down and treated by PI3K activator. ( a ) The selection of BCAR3 siRNA sequence. BCAR3 mRNA expression in KTC-1 cells after transfection with three different siRNAs (si BCAR3 -605, si BCAR3 -981 and si BCAR3 -1221) detected by quantitative real-time PCR. Statistical comparisons between each siRNA group and the negative control (si-NC) group were performed using unpaired t -tests. ( b , c ) Validation of BCAR3 knockdown efficiency. BCAR3 mRNA expression in TPC-1 ( b ) and KTC-1 ( c ) cells after siRNA-mediated knockdown detected by quantitative real-time PCR (n = 3). GAPDH was used as an internal control. Statistical significance was determined by an unpaired t -test. ( d , e ) Expression of phosphorylated mTOR (pmTOR), total mTOR, phosphorylated AKT (pAKT), total AKT, BCAR3 in TPC-1 ( d ) and KTC-1 ( e ) cells was detected by Western blot. β-actin was used as a loading control. The corresponding quantitative analysis of protein expression were shown in the bar graphs below. Statistical comparisons between specified groups (KD vs. NC; AC vs. KD) for each protein were performed using unpaired t -tests. Data in all bar graphs are presented as mean with SD; * p < 0.05, ** p < 0.01, **** p < 0.001. The uncropped blots are shown in .

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Knockdown, Negative Control, Selection, Sequencing, Expressing, Transfection, Real-time Polymerase Chain Reaction, Biomarker Discovery, Control, Western Blot

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: Effects of BCAR3 knockdown and PI3K activation on cell proliferation and colony formation in thyroid cancer cell lines. The abbreviations of the four groups in different cells are as follows: WT, wild type; NC, negative control with si-NC treated; KD, knockdown by si- BCAR3 ; AC, knocked down and treated by PI3K activator. ( a , b ) Proliferation of TPC-1 ( a ) and KTC-1 ( b ) cells was measured using CCK-8 assay (n = 6 biological replicates). Data are presented as mean ± SD. Statistical comparisons between KD vs. NC, and AC vs. KD groups were assessed using unpaired t -tests ** p < 0.01, *** p < 0.005, **** p < 0.001, KD vs. NC; # p < 0.05, ### p < 0.005, #### p < 0.001, AC vs. KD. ( c , d ) Representative images of colony formation assays in TPC-1 ( c ) and KTC-1 ( d ) cells under the same four conditions (n = 3 biological replicates). ( e , f ) Quantification of colony formation efficiency in TPC-1 ( e ) and KTC-1 ( f ) cells. Data are presented as mean with SD. Statistical comparisons between KD vs. NC and AC vs. KD groups were performed using unpaired t -tests. * p < 0.05, *** p < 0.005, **** p < 0.001.

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Knockdown, Activation Assay, Negative Control, CCK-8 Assay

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: Effects of BCAR3 knockdown and PI3K activation on cell migration and invasion in TPC-1 and KTC-1 thyroid cancer cell lines. The four experimental groups were: WT, wild type; NC, negative control with si-NC treated; KD, knockdown by si- BCAR3 ; AC, knocked down and treated by PI3K activator. ( a , b ) Cell migration in TPC-1 ( a ) and KTC-1 ( b ) cells was examined by wound healing assay. Quantitative analysis of the relative wound closure area is shown on the right (n = 6 biological replicates). Data are presented as mean with SD. ( c , d ) Cell migration and invasion in TPC-1 ( c ) and KTC-1 ( d ) cells was evaluated by transwell assay. Quantitative data on the number of migrated or invaded cells are shown on the right (n = 3 biological replicates). Data are presented as mean with SD. Statistical comparisons between KD vs. NC and AC vs. KD groups were performed using unpaired t -tests. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Knockdown, Activation Assay, Migration, Negative Control, Wound Healing Assay, Transwell Assay

Journal: Cancers

Article Title: BCAR3 Hypomethylation as a Potential Diagnostic Marker for Thyroid Cancer and Its Mechanism via Promoting EMT and AKT/mTOR Pathway

doi: 10.3390/cancers18020267

Figure Lengend Snippet: Effects of BCAR3 knockdown on epithelial–mesenchymal transition (EMT) markers in TPC-1 and KTC-1 cells. The abbreviations of the four groups in different cells are as follows: WT, wild type; NC, negative control with si-NC treated; KD, knockdown by si- BCAR3 ; AC, knocked down and treated by PI3K activator. ( a , b ) Expression of BCAR3, E-cadherin, N-cadherin, and Vimentin in TPC-1 ( a ) and KTC-1 ( b ) cells across four conditions—wild-type (WT), negative control (NC), BCAR3 knockdown (KD), and BCAR3 knockdown with PI3K activator (AC)—was analyzed by Western blot. Quantification of relative protein band intensity is shown below the blots (n = 3 biological replicates). Data are presented as mean with SD. ( c ) The localization of E-cadherin (green) and Vimentin (red) in KTC-1 cells under the same conditions as in ( a , b ) was examined by immunofluorescence staining. Nuclei are stained with Hoechst (blue). The bar graph quantifies the mean fluorescence intensity of E-cadherin and Vimentin (n = 4 biological replicates). Data are presented as mean with SD. For all quantitative analyses, statistical comparisons between specific groups (KD vs. NC; AC vs. KD) were performed using unpaired t -tests. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. The uncropped blots are shown in .

Article Snippet: After peroxidase blocking with 3% H 2 O 2 , sections were incubated with 5% BSA and then with anti-BCAR3 antibody (1:100, Abmart, TD3048, Shanghai, China) at 4 °C overnight.

Techniques: Knockdown, Negative Control, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Boron Triggers Hepatic Ferroptosis: Unveiling the Dual-Pathogenic Nexus of Oxidative Stress and SLC7A11/GPX4 Dysregulation

doi: 10.3390/ani16050832

Figure Lengend Snippet: B-induced ferroptosis and ferritinophagy in chicken livers. ( A ) Hepatic total iron, Fe 2+ , and Fe 3+ content. ( B – G ) The relative mRNA expression of SLC7A11 , GPX4 , ACSL4 , TF , TFR , and FTH1 in liver. ( H – J ) Quantification of Western blots and representative Western blotting of SLC7A11, GPX4, FTH1, FTL, TF, and TFR in liver of chicken of B treatment.

Article Snippet: The primary antibodies used included β-actin (1:1500, Servicebio, Wuhan, China), GAPDH (1:20,000, Proteintech, Wuhan, China), Nrf2 (1:2000, Proteintech, Wuhan, China), Keap1 (1:5000, Proteintech, Wuhan, China), ACSL4 (1:30,000, Proteintech, Wuhan, China), HO-1 (1:3000, Affinity, Jiangsu, China), CAT (1:1500, Affinity, Jiangsu, China), GPX4 (1:1000, Bioworld, Nanjing, China), SLC7A11 (1:1000, Bioss, Beijing, China), FTH1 (1:800, Wanleibio, Shenyang, China), TF (1:1000, Wanleibio, Shenyang, China), TFR1 (1:800, Wanleibio, Shenyang, China), and FTL (1:2000, Abmart, Shanghai, China).

Techniques: Expressing, Western Blot