rabbit monoclonal anti hsp90β antibody  (Danaher Inc)


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    Danaher Inc rabbit monoclonal anti hsp90β antibody
    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and <t>HSP90</t> enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres
    Rabbit Monoclonal Anti Hsp90β Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti hsp90β antibody/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal anti hsp90β antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    Journal: Cellular and Molecular Life Sciences: CMLS

    doi: 10.1007/s00018-023-05046-6

    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres
    Figure Legend Snippet: Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres

    Techniques Used: Expressing, Whisker Assay, Two Tailed Test, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Binding Assay, Reverse Transcription Polymerase Chain Reaction

    Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
    Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
    Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Techniques Used: Binding Assay

    rabbit monoclonal anti hsp90β antibody  (Danaher Inc)


    Bioz Verified Symbol Danaher Inc is a verified supplier
    Bioz Manufacturer Symbol Danaher Inc manufactures this product  
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  • 86

    Structured Review

    Danaher Inc rabbit monoclonal anti hsp90β antibody
    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and <t>HSP90</t> enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres
    Rabbit Monoclonal Anti Hsp90β Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti hsp90β antibody/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal anti hsp90β antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    Journal: Cellular and Molecular Life Sciences: CMLS

    doi: 10.1007/s00018-023-05046-6

    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres
    Figure Legend Snippet: Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres

    Techniques Used: Expressing, Whisker Assay, Two Tailed Test, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Binding Assay, Reverse Transcription Polymerase Chain Reaction

    Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
    Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
    Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Techniques Used: Binding Assay

    rabbit monoclonal antibody against ionized calcium binding adaptor molecule 1  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against ionized calcium binding adaptor molecule 1
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; <t>Iba1:</t> ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Rabbit Monoclonal Antibody Against Ionized Calcium Binding Adaptor Molecule 1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression"

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.390960

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Figure Legend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Techniques Used: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Immunofluorescence

    rabbit monoclonal antibody against acsl4  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against acsl4
    Rabbit Monoclonal Antibody Against Acsl4, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit monoclonal antibody against inducible nitric oxide synthase  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against inducible nitric oxide synthase
    Rabbit Monoclonal Antibody Against Inducible Nitric Oxide Synthase, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal antibody against inducible nitric oxide synthase/product/Danaher Inc
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    rabbit monoclonal antibody against hmox1  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against hmox1
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; <t>HMOX1:</t> heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Rabbit Monoclonal Antibody Against Hmox1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal antibody against hmox1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal antibody against hmox1 - by Bioz Stars, 2024-06
    86/100 stars

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    1) Product Images from "Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression"

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.390960

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Figure Legend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Techniques Used: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Sequences and titer information of lentiviruses
    Figure Legend Snippet: Sequences and titer information of lentiviruses

    Techniques Used:

    Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.
    Figure Legend Snippet: Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.

    Techniques Used:

    Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.
    Figure Legend Snippet: Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.

    Techniques Used: Expressing, Functional Assay, Binding Assay

    SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Expressing, Transmission Assay, Electron Microscopy

    Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Western Blot, Expressing

    Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Immunofluorescence

    Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining

    rabbit monoclonal antibody against hmox1  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against hmox1
    Rabbit Monoclonal Antibody Against Hmox1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit monoclonal antibody against hmox1  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against hmox1
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; <t>HMOX1:</t> heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Rabbit Monoclonal Antibody Against Hmox1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal antibody against hmox1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal antibody against hmox1 - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression"

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.390960

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Figure Legend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Techniques Used: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Sequences and titer information of lentiviruses
    Figure Legend Snippet: Sequences and titer information of lentiviruses

    Techniques Used:

    Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.
    Figure Legend Snippet: Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.

    Techniques Used:

    Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.
    Figure Legend Snippet: Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.

    Techniques Used: Expressing, Functional Assay, Binding Assay

    SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Expressing, Transmission Assay, Electron Microscopy

    Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Western Blot, Expressing

    Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Immunofluorescence

    Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining

    rabbit monoclonal antibody against acsl4  (Danaher Inc)


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    Danaher Inc rabbit monoclonal antibody against acsl4
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; <t>ACSL4:</t> acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Rabbit Monoclonal Antibody Against Acsl4, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal antibody against acsl4/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal antibody against acsl4 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression"

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.390960

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
    Figure Legend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Techniques Used: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Primer sequences used in PCR analysis
    Figure Legend Snippet: Primer sequences used in PCR analysis

    Techniques Used: Sequencing

    Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.
    Figure Legend Snippet: Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.

    Techniques Used: Expressing, Functional Assay, Binding Assay

    SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Staining, Expressing, Transmission Assay, Electron Microscopy

    Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Met attenuates neuronal cell ferroptosis and neuroinflammation after SCI. (A–D) Representative IF staining images showing GPX4 + and ACSL4 + neurons, and quantitative analysis of the results. Ferroptosis in the SCI group was increased compared with the sham group, and this effect was dramatically reversed by Met treatment ( n = 5). (E–H) Representative IF staining images showing IL-1β + and IL-6 + neurons, and quantitative analysis of the results. Neuroinflammation in the SCI group was increased compared with the sham group, and this effect was dramatically reversed by Met treatment ( n = 5). Red indicates Cy3-positive neurons, and green indicates cells that stained positively for GPX4, ACSL4, IL-1β, or IL-6. Scale bars: 50 μm. The data are expressed as the mean ± SEM. *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). ACSL4: Acyl-coenzyme A synthetase long-chain family member 4; GPX4: glutathione peroxidase 4; IF: immunofluorescence; IL-1β: interleukin 1β; IL-6: interleukin 6; Met: metformin; SCI: spinal cord injury.
    Figure Legend Snippet: Met attenuates neuronal cell ferroptosis and neuroinflammation after SCI. (A–D) Representative IF staining images showing GPX4 + and ACSL4 + neurons, and quantitative analysis of the results. Ferroptosis in the SCI group was increased compared with the sham group, and this effect was dramatically reversed by Met treatment ( n = 5). (E–H) Representative IF staining images showing IL-1β + and IL-6 + neurons, and quantitative analysis of the results. Neuroinflammation in the SCI group was increased compared with the sham group, and this effect was dramatically reversed by Met treatment ( n = 5). Red indicates Cy3-positive neurons, and green indicates cells that stained positively for GPX4, ACSL4, IL-1β, or IL-6. Scale bars: 50 μm. The data are expressed as the mean ± SEM. *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). ACSL4: Acyl-coenzyme A synthetase long-chain family member 4; GPX4: glutathione peroxidase 4; IF: immunofluorescence; IL-1β: interleukin 1β; IL-6: interleukin 6; Met: metformin; SCI: spinal cord injury.

    Techniques Used: Staining, Immunofluorescence

    HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.
    Figure Legend Snippet: HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Techniques Used: Western Blot, Expressing

    alexa fluor 647 rabbit monoclonal anti neun antibody  (Danaher Inc)


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    Danaher Inc alexa fluor 647 rabbit monoclonal anti neun antibody
    On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.
    Alexa Fluor 647 Rabbit Monoclonal Anti Neun Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation"

    Article Title: Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.390953

    On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.
    Figure Legend Snippet: On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.

    Techniques Used: Immunofluorescence, Fluorescence, Positive Control

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    Danaher Inc rabbit monoclonal anti hsp90β antibody
    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and <t>HSP90</t> enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres
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    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; <t>Iba1:</t> ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
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    Danaher Inc rabbit monoclonal antibody against acsl4
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; <t>Iba1:</t> ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
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    Danaher Inc rabbit monoclonal antibody against inducible nitric oxide synthase
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; <t>Iba1:</t> ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
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    Danaher Inc rabbit monoclonal antibody against hmox1
    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; <t>HMOX1:</t> heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.
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    Danaher Inc alexa fluor 647 rabbit monoclonal anti neun antibody
    On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.
    Alexa Fluor 647 Rabbit Monoclonal Anti Neun Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Stabilization of MAPK12-G4 motifs by TGS24 enhances stemness-markers via HSP90-RAS-c-JUN-NANOG axis. A Flow cytometric analyses of CD44 and CD24 expression in MDAMB-231 spheres upon TGS24 (50 nM) treatment for 24 h. Box and whisker plot displays the percentage of CD44 low/CD24low and CD44high /CD24 low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). B Violin plot showing MAPK12 expression in CD44high /CD24low population upon TGS24 (50 nM) treatment for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). C Western blot analyses of the proteins in RAS pathway: RAS, c-JUN, HSP90, MAPK12 under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. GAPDH considered as housekeeping protein. D Co-immunoprecipitation analysis showing tetrameric complex of RAS, HSP90, MAPK12, and c-JUN. MDAMB-231 spheres that express RAS, are subjected to immunoprecipitation (IP) with anti-RAS antibody and to immunoblotting (IB) with anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies upon TGS24 (50 nM) treatment for 24 h. E ChIP analyses. ChIP results using anti-RAS, anti-HSP90, anti-c-JUN, and anti-MAPK12 antibodies suggesting MAPK12, c-JUN, RAS, and HSP90 enrichment at NANOG promoter under TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. F Schematic representation of NANOG promoter region (AP1 site). Transcription initiation site is marked with an arrow. Position of the ChIP primers used in semi-quantitative and Real-time PCR reactions following immunoprecipitation is indicated. Input fraction indicates total DNA; negative control immunoprecipitation uses rabbit IgG showing no signal. G Quantification of MAPK12, RAS, c-JUN, and HSP90 binding at NANOG promoter by percentage of input method based on quantitative real-time PCR under aforementioned conditions. Error bars represent mean ± SE (N = 3). Statistical differences are determined by one-way ANOVA followed by Tukey–Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001). H RT-PCR analyses. Expression profile of OCT-4, SOX2, and NANOG transcripts upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres. I Quantification of the transcripts’ level of OCT-4, SOX2, and NANOG relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). J Western blot analyses of OCT-4, SOX2, CD-44, and NANOG upon the treatment of TGS24 (50 nM) treatment for 24 h in MDAMB-231 spheres

    Article Snippet: Detection of HSP90, c-JUN, and MAPK12, immunoprecipitation with Rabbit monoclonal anti-Hsp90β antibody (Abcam), Anti c-JUN Rabbit polyclonal antibody (Abcam), Rabbit anti-p38γ-antibody (CST) in the IP buffer (20 mM Tris – HCl (pH 8.0), 0.2% Nonidet P-40, 1 mM dithiothreitol, and the protease inhibitory cocktail) followed by washing in the wash buffer that contains 1% Nonidet P-40.

    Techniques: Expressing, Whisker Assay, Two Tailed Test, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Binding Assay, Reverse Transcription Polymerase Chain Reaction

    Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Article Snippet: Detection of HSP90, c-JUN, and MAPK12, immunoprecipitation with Rabbit monoclonal anti-Hsp90β antibody (Abcam), Anti c-JUN Rabbit polyclonal antibody (Abcam), Rabbit anti-p38γ-antibody (CST) in the IP buffer (20 mM Tris – HCl (pH 8.0), 0.2% Nonidet P-40, 1 mM dithiothreitol, and the protease inhibitory cocktail) followed by washing in the wash buffer that contains 1% Nonidet P-40.

    Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Article Snippet: Detection of HSP90, c-JUN, and MAPK12, immunoprecipitation with Rabbit monoclonal anti-Hsp90β antibody (Abcam), Anti c-JUN Rabbit polyclonal antibody (Abcam), Rabbit anti-p38γ-antibody (CST) in the IP buffer (20 mM Tris – HCl (pH 8.0), 0.2% Nonidet P-40, 1 mM dithiothreitol, and the protease inhibitory cocktail) followed by washing in the wash buffer that contains 1% Nonidet P-40.

    Techniques: Binding Assay

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Article Snippet: The primary antibodies used for immunofluorescence were rabbit monoclonal antibody against GPX4 (1:500, Abcam, Cat# ab125066, RRID: AB_10973901), rabbit monoclonal antibody against ACSL4 (1:500, Abcam, Cat# ab155282, RRID: AB_2714020), rabbit polyclonal antibody against interleukin-1β (1:200, Affinity, Cat# AF5103, RRID: AB_2837589), rabbit polyclonal antibody against interleukin-6 (1:200, Affinity, Cat# DF6087, RRID: AB_2838055), rabbit polyclonal antibody against neuronal nuclei (1:2000, Servicebio, Cat# GB11138, RRID: AB_2868432), rabbit monoclonal antibody against ionized calcium binding adaptor molecule 1 (Iba1; 1:3000, Abcam, Cat# ab178846, RRID: AB_263685), rabbit monoclonal antibody against inducible nitric oxide synthase (iNOS, 1:300, Abcam, Cat# ab178945, RRID: AB_2861417), and rabbit polyclonal antibody against arginase 1 (Arg1, 1:300, GeneTex, San-Antonio, TX, USA, Cat# GTX109242, RRID: AB_2036264).

    Techniques: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The primary antibodies used for immunofluorescence were rabbit monoclonal antibody against GPX4 (1:500, Abcam, Cat# ab125066, RRID: AB_10973901), rabbit monoclonal antibody against ACSL4 (1:500, Abcam, Cat# ab155282, RRID: AB_2714020), rabbit polyclonal antibody against interleukin-1β (1:200, Affinity, Cat# AF5103, RRID: AB_2837589), rabbit polyclonal antibody against interleukin-6 (1:200, Affinity, Cat# DF6087, RRID: AB_2838055), rabbit polyclonal antibody against neuronal nuclei (1:2000, Servicebio, Cat# GB11138, RRID: AB_2868432), rabbit monoclonal antibody against ionized calcium binding adaptor molecule 1 (Iba1; 1:3000, Abcam, Cat# ab178846, RRID: AB_263685), rabbit monoclonal antibody against inducible nitric oxide synthase (iNOS, 1:300, Abcam, Cat# ab178945, RRID: AB_2861417), and rabbit polyclonal antibody against arginase 1 (Arg1, 1:300, GeneTex, San-Antonio, TX, USA, Cat# GTX109242, RRID: AB_2036264).

    Techniques: Staining, Immunofluorescence

    Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Experimental flowchart for the in vivo experiments. 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; Arg1: arginase 1; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; IF: immunofluorescence; IHC: immunohistochemistry; Iba1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; iNOS: inducible nitric oxide synthase; LV: lentivirus; PCR: polymerase phain peaction; SCI: spinal cord injury; TEM: transmission electron microscopy; WB: western blotting.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: In Vivo, Immunofluorescence, Immunohistochemistry, Binding Assay, Transmission Assay, Electron Microscopy, Western Blot

    Sequences and titer information of lentiviruses

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Sequences and titer information of lentiviruses

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques:

    Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Hmox1 is significantly upregulated in a rat model of SCI. (A) Common DEGs at five time points in the GSE45006 dataset. (B) Ferroptosis-related DEGs (from the FerrDb V2 database (http://www.zhounan.org/ferrdb/current/)) in the GSE45006 dataset. (C) Ferroptosis-related DEGs in the GSE2599 dataset. (D) Common ferroptosis-related DEGs in the GSE45006 and GSE2599 datasets. (E–I) Visualization of DEGs at different time points in the GSE45006 dataset, including three common ferroptosis-related DEGs. (J) Visualization of DEGs in the GSE2599 dataset, containing three common ferroptosis-related DEGs. |logFC| > 1, adjusted P value < 0.05. DEGs: Differentially expressed genes; dpi: day(s) post injury; FC: fold change; Gch1 : GTP cyclohydrolase 1 gene; Hmox1 : heme oxygenase-1; SCI: spinal cord injury; Tgfbr1 : transforming growth factor beta receptor 1.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques:

    Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Dynamic changes in the expression of inflammatory cytokine genes and ferroptosis-related genes (FRGs) after SCI. (A, B) Functional annotation and pathway enrichment analysis of 17 ferroptosis-related DEGs from the GSE45006 dataset. Red represents molecules, and blue represents GO and KEGG items. (C) Protein-protein interaction network of 17 ferroptosis-related DEGs. (D–L) Dynamic changes in nine FRGs of interest after SCI. Red line: baseline value, indicating that the expression level of this gene at this time point was 1 time that of the sham group (2 0 ). Blue line: |logFC| = 1, indicating that the expression level of this gene at this time point was 2 time that of the sham group (2 1 ). Adjusted * P < 0.05, adjusted *** P < 0.001 (independent samples t test). Acsl4 : Acyl-coenzyme A synthetase long-chain family member 4; Atf3 : activating transcription factor 3; BP: biological brocess; CC: cell component; CD44 : cluster of differentiation 44; Cybb : cytochrome b-245 beta chain; DEGs: differentially expressed genes; FRG: ferroptosis-related gene; Gpx4 : glutathione peroxidase 4; HIF-1 : hypoxia inducible factor-1; Hmox1 : heme oxygenase-1; Il-10 : interleukin 10; Il-1 β: interleukin 1β; Il-6 : interleukin 6; KEGG: Kyoto Encyclopedia of Genes and Genomes; Lpcat3 : lysophosphatidylcholine acyltransferase 3; Lrrfip1 : lrr binding flii interacting protein 1; MF: molecular function; Ripk1 : receptor-interacting protein kinase 1; SCI: spinal cord injury; Slc3a2 : solute carrier family 3 member 2; Slc7a11 : solute carrier family 7 member 11; Stat3 : signal transducer and activator of transcription 3; Tgfbr1 : transforming growth factor beta receptor 1; Tlr4 : Toll-like receptor 4.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Expressing, Functional Assay, Binding Assay

    SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: SCI-induced lipid peroxidation and iron deposition aggravate ferroptosis. (A) Schematic diagrams of the sham, 1 dpi, and 7 dpi groups and the main region of interest (ROI). (B) Representative area of interest in the ROI. (C, D) Representative images of Prussian blue staining and quantification of iron-positive cells. Iron deposition was not detected until 3 days after SCI and was significantly elevated on days 7 and 14 compared with the sham group ( n = 6). Scale bars: 50 μm. (E, F) Representative images of Fluoro-Jade B (FJB) staining and quantification of FJB-positive neurons. The number of degenerating neurons was significantly increased on day 3, but not on days 7 and 14, after SCI compared with the sham group ( n = 6). Scale bars: 50 μm. (G–J) Relative protein expression levels of 4HNE, ACSL4, GPX4, and HMOX1, normalized to the sham group ( n = 5). (K) Mitochondrial ultrastructure was examined by transmission electron microscopy ( n = 3). Scale bars: 2 μm (upper panel), 500 nm (lower panel). Black arrows: normal mitochondria, red arrows: damaged mitochondria, yellow arrows: smaller mitochondria (that did not exhibit shrunken morphology). The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; hpi: hours post-injury; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Staining, Expressing, Transmission Assay, Electron Microscopy

    Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Met treatment increases HMOX1 expression after SCI. (A, B) Representative images of immunohistochemistry (IHC) staining and quantification of HMOX1 expression in the gray matter. HMOX1 expression was significantly upregulated in the first 7 days after injury in the SCI group compared with the sham group ( n = 6). Scale bars: 50 μm. (C, D) Representative images of IHC staining and quantification of HMOX1 expression in the white matter. HMOX1 expression was significantly upregulated in the first 14 days after injury in the SCI group compared with the sham group. HMOX1 expression was highest on day 3 after SCI compared with the Sham group ( n = 6). Red arrows: positively stained cells, black arrows: non-stained cells. Scale bars: 50 μm. (E–G) Relative mRNA levels of Gpx4 , Acsl4 , and Hmox1 were determined by PCR ( n = 6). (H–J) Relative protein expression levels of ACSL4, HMOX1, and 4HNE in the indicated groups ( n = 5). Expression levels shown in E–G were normalized to those in the sham group. The data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni post hoc test (B, D) or two-way analysis of variance with Tukey's post hoc test (E–J)). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; Met: metformin; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Expressing, Immunohistochemistry, Staining

    HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: HMOX1 knockdown partially reverses the anti-ferroptotic effects of Met after SCI. (A–D) Representative western blot showing HMOX1, 4HNE, ACSL4, and GPX4 expression levels in the indicated groups at 3 dpi ( n = 5). Expression levels were normalized to the β-actin expression level in the same lane. The data are expressed as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). 4HNE: 4-Hydroxynonenal; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; dpi: day(s) post injury; GPX4: glutathione peroxidase 4; HMOX1: heme oxygenase-1; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Western Blot, Expressing

    Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Met promotes the polarization of M1-type microglia/macrophages towards an M2 phenotype in an HMOX1-independent manner. (A, B) Representative IF staining images and the proportion of iNOS + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M1-type to M2-type microglia in the SCI group was increased compared with that in the sham group, and this effect was dramatically reversed by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. (C, D) Representative IF staining images and the proportion of Arg1 + -stained microglia/macrophages in the indicated groups at 3 dpi. In both the NC and KD groups, the ratio of M2-type to M1-type microglia in the SCI group was increased compared with that in the sham group, and this ratio was further increased by Met treatment. After HMOX1 knockdown, the ratio in the SCI and Met subgroups of the KD group was lower than that in the NC group ( n = 5). Red indicates Iba1 (stained with Cy3), and green indicates iNOS or Arg1 (stained with Alexa Fluor 488). Scale bars: 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). Arg1: Arginase 1; dpi: day(s) post injury; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Staining, Immunofluorescence

    Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Metformin alleviates spinal cord injury by inhibiting nerve cell ferroptosis through upregulation of heme oxygenase-1 expression

    doi: 10.4103/1673-5374.390960

    Figure Lengend Snippet: Met exerts a neuroprotective effect in an HMOX1-dependent manner. (A, B) Representative FJB staining images and the proportion of FJB + neurons at 3 dpi. In both the NC and KD groups, the ratio of FJB + neurons to unstained neurons in the SCI group was increased compared with that in the sham group. This effect was dramatically reversed by Met treatment in the NC group but not in the KD group. After HMOX1 knockdown, the ratio in the SCI subgroup and the Met subgroup of the KD group was higher than that in the NC group ( n = 5). Scale bars: 50 μm. ** P < 0.01, *** P < 0.001 (two-way analysis of variance with Tukey's post hoc test). dpi: Day(s) post injury; FJB: Fluoro-Jade B; KD: knockdown; Met: metformin; NC: normal control; ns: not significant; SCI: spinal cord injury.

    Article Snippet: The slides were then incubated with primary rabbit monoclonal antibody against HMOX1 (1:20,000, Abcam, Cat# ab189491, RRID: AB_1267209) at 4°C overnight in a humid box.

    Techniques: Staining

    On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.

    Journal: Neural Regeneration Research

    Article Title: Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation

    doi: 10.4103/1673-5374.390953

    Figure Lengend Snippet: On day 21 after injury, the promoting effect of BA on neuronal autophagy in the spinal cord tissue of SCI rats was observed. (A, B) Representative immunofluorescence images (A, original magnification 400×) and statistical analysis (B) of LC3 fluorescence intensity. LC3 fluorescence intensity (green Alexa Fluor® 488) was decreased in neurons (marked by NeuN, red, Alexa Fluor® 647) in the model group compared with that in the sham group; BA and methylprednisolone treatment enhanced LC3 fluorescence intensity (green, Alexa Fluor® 488). Scale bars: 100 μm. (C, D) Representative immunofluorescence images (C, original magnification 400×) and statistical analysis of P62 fluorescence intensity (D). Scale bars: 100 μm. P62 fluorescence intensity (green, Alexa Fluor® 488) in neurons (marked by NeuN, red, Alexa Fluor® 647) was stronger in the model group than in the sham group; BA and methylprednisolone treatment decreased P62 fluorescence intensity (green Alexa Fluor® 488). Data are expressed as mean ± SD ( n = 6). ** P < 0.01, vs. sham group; ## P < 0.01, vs . model group (one-way analysis of variance followed by Tukey's post hoc test). BA: Biochanin A; DAPI: 4,6-diamino-2-phenylindole; LC3: microtube-associated protein 1 light-chain 3; P62: sequestosome-1; PC: positive control; SCI: spinal cord injury.

    Article Snippet: Each slice was blocked in 5% bovine serum albumin for 60 minutes at 37°C, then stained with Alexa Fluor® 488 anti-microtubule-associated protein light chain 3 (LC3) rabbit monoclonal antibody (1:50, CST, Danvers, MA, USA, Cat# 13082, RRID: AB_2687880), anti-SQSTM1/p62 rabbit polyclonal antibody (1:100, Affinity, Cincinnati, OH, USA Cat# AF5384, RRID: AB_2837869), and Alexa Fluor® 647 rabbit monoclonal anti-NeuN antibody (1:50, Abcam, Cambridge, UK, Cat# ab190565, RRID: AB_2732785) overnight at 4°C.

    Techniques: Immunofluorescence, Fluorescence, Positive Control