anti hnrnp k monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti hnrnp k monoclonal antibody
    Expression patterns of <t>hnRNP</t> <t>K</t> in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.
    Anti Hnrnp K Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hnrnp k monoclonal antibody/product/Cell Signaling Technology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hnrnp k monoclonal antibody - by Bioz Stars, 2023-02
    92/100 stars

    Images

    1) Product Images from "Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma"

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    Journal: Oncology Letters

    doi: 10.3892/ol.2020.12257

    Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.
    Figure Legend Snippet: Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining

    hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.
    Figure Legend Snippet: hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.

    Techniques Used: Migration, Transfection, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Colony Assay, Expressing, Wound Healing Assay, Transwell Assay, Negative Control

    hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.
    Figure Legend Snippet: hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.

    Techniques Used: In Vivo, Derivative Assay, Transfection, Injection, shRNA, Negative Control

    GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.
    Figure Legend Snippet: GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.

    Techniques Used: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Plasmid Preparation, Inhibition, Quantitative RT-PCR, Small Interfering RNA, Negative Control

    anti hnrnp k monoclonal antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 92

    Structured Review

    Cell Signaling Technology Inc anti hnrnp k monoclonal antibody
    Expression patterns of <t>hnRNP</t> <t>K</t> in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.
    Anti Hnrnp K Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hnrnp k monoclonal antibody/product/Cell Signaling Technology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hnrnp k monoclonal antibody - by Bioz Stars, 2023-02
    92/100 stars

    Images

    1) Product Images from "Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma"

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    Journal: Oncology Letters

    doi: 10.3892/ol.2020.12257

    Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.
    Figure Legend Snippet: Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining

    hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.
    Figure Legend Snippet: hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.

    Techniques Used: Migration, Transfection, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Colony Assay, Expressing, Wound Healing Assay, Transwell Assay, Negative Control

    hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.
    Figure Legend Snippet: hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.

    Techniques Used: In Vivo, Derivative Assay, Transfection, Injection, shRNA, Negative Control

    GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.
    Figure Legend Snippet: GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.

    Techniques Used: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Plasmid Preparation, Inhibition, Quantitative RT-PCR, Small Interfering RNA, Negative Control

    mouse monoclonal anti hnrnp k  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse monoclonal anti hnrnp k
    Association of nuclear and cytoplasmic <t> hnRNP K </t> expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.
    Mouse Monoclonal Anti Hnrnp K, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti hnrnp k/product/Cell Signaling Technology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti hnrnp k - by Bioz Stars, 2023-02
    92/100 stars

    Images

    1) Product Images from "Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma"

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2020.4718

    Association of nuclear and cytoplasmic  hnRNP K  expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.
    Figure Legend Snippet: Association of nuclear and cytoplasmic hnRNP K expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.

    Techniques Used: Expressing

    (A) Representative images of HNSCC tissue samples for hnRNP K IHC staining focusing on nuclear (nuc) and cytoplasmic (cyt) hnRNP K expression (scale bar, 200 μ m): hnRNP K-negative (upper panel), nuclear hnRNP K expression (middle panel) and hnRNP K nuclear and cytoplasmic expression (lower panel). (B) Representative images of p16 INK4A-negative (upper panel) and -positive (lower panel) HNSCC samples. Staining for p16INK4A served as a surrogate marker for HPV-positive HNSCC. HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; IHC, immunohistochemistry; HPV, human papillomavirus.
    Figure Legend Snippet: (A) Representative images of HNSCC tissue samples for hnRNP K IHC staining focusing on nuclear (nuc) and cytoplasmic (cyt) hnRNP K expression (scale bar, 200 μ m): hnRNP K-negative (upper panel), nuclear hnRNP K expression (middle panel) and hnRNP K nuclear and cytoplasmic expression (lower panel). (B) Representative images of p16 INK4A-negative (upper panel) and -positive (lower panel) HNSCC samples. Staining for p16INK4A served as a surrogate marker for HPV-positive HNSCC. HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; IHC, immunohistochemistry; HPV, human papillomavirus.

    Techniques Used: Immunohistochemistry, Expressing, Staining, Marker

    Association of  hnRNP K  IHC score with the analyzed categories (tissue, sex and stage) among 117 HNSCC cases and 15 normal oral tissue samples.
    Figure Legend Snippet: Association of hnRNP K IHC score with the analyzed categories (tissue, sex and stage) among 117 HNSCC cases and 15 normal oral tissue samples.

    Techniques Used:

    (A) Representative images of HNSCC cell clusters stained for p16INK4a using immunofluorescence illustrate the HPV status of Cal-27 (HPV-negative) and UPCI-SCC 154 (HPV-positive) cells (scale bar, 40 μ m). (B) Clonogenic survival assay of the HNSCC cell lines Cal-27 and UPCI-SCC 154 after 9 days. Data are presented as mean ± SD of 4 independent experiments. (C) Representative immunoblots demonstrate a rapid increase in cellular hnRNP K levels induced by IR (2 Gy), reaching maximum levels after 30-60 min before normalization of cellular hnRNP K levels within 24 h. Ratios represent hnRNP K/GAPDH referenced to non-irradiated control. (D) Dose-dependent accumulation of cellular hnRNP K 1 h after IR. (E) Immunofluorescence microscopy indicated cytoplasmic hnRNP K accumulation 1 h after irradiating cells with 2 Gy (scale bar, 20 μ m). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; HPV, human papillomavirus; IR, ionizing radiation.
    Figure Legend Snippet: (A) Representative images of HNSCC cell clusters stained for p16INK4a using immunofluorescence illustrate the HPV status of Cal-27 (HPV-negative) and UPCI-SCC 154 (HPV-positive) cells (scale bar, 40 μ m). (B) Clonogenic survival assay of the HNSCC cell lines Cal-27 and UPCI-SCC 154 after 9 days. Data are presented as mean ± SD of 4 independent experiments. (C) Representative immunoblots demonstrate a rapid increase in cellular hnRNP K levels induced by IR (2 Gy), reaching maximum levels after 30-60 min before normalization of cellular hnRNP K levels within 24 h. Ratios represent hnRNP K/GAPDH referenced to non-irradiated control. (D) Dose-dependent accumulation of cellular hnRNP K 1 h after IR. (E) Immunofluorescence microscopy indicated cytoplasmic hnRNP K accumulation 1 h after irradiating cells with 2 Gy (scale bar, 20 μ m). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; HPV, human papillomavirus; IR, ionizing radiation.

    Techniques Used: Staining, Immunofluorescence, Clonogenic Cell Survival Assay, Western Blot, Irradiation, Microscopy

    (A) Effective hnRNP K knockdown by transient transfection was verified by immunoblotting. Mock transfection served as control. (B) Representative images of colonies after 9 days of incubation. (C) Statistical analysis of clonogenic survival assays during hnRNP K knockdown. All experiments were carried out in quadruplicate. Data are presented as mean ± SD. * P<0.05 (Kruskal Wallis test, Tukey's post hoc test). (D) ELISA showed significantly increased levels of cellular active caspase-3 in Cal-27 and UPCI-SCC-154 cells parallel to hnRNP K knockdown. Data are presented as mean ± SD. * P<0.05; n.s., not significant; n=6 (Kruskal Wallis test, Holm-Sidak post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K.
    Figure Legend Snippet: (A) Effective hnRNP K knockdown by transient transfection was verified by immunoblotting. Mock transfection served as control. (B) Representative images of colonies after 9 days of incubation. (C) Statistical analysis of clonogenic survival assays during hnRNP K knockdown. All experiments were carried out in quadruplicate. Data are presented as mean ± SD. * P<0.05 (Kruskal Wallis test, Tukey's post hoc test). (D) ELISA showed significantly increased levels of cellular active caspase-3 in Cal-27 and UPCI-SCC-154 cells parallel to hnRNP K knockdown. Data are presented as mean ± SD. * P<0.05; n.s., not significant; n=6 (Kruskal Wallis test, Holm-Sidak post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K.

    Techniques Used: Transfection, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

    Knockdown of hnRNP K inhibits growth of HNSSC xenografts on the chick egg CAM in vivo . Cells (mock or siRNA, ± irradiation) were seeded on the CAM of fertilized chick eggs 7 days after the start of incubation (1.5×10 6 cells/egg in medium/Matrigel 1:1). After an incubation period of 4 days at 37°C, the tumors were collected, imaged, fixed and embedded in paraffin for immunohistochemical analysis. Sections (5 μ m) were stained for hnRNP K, proliferation marker Ki-67 and the angiogenesis marker desmin. Each group contained 9-10 tumor-bearing eggs. (A and B) Representative images of tumor xenografts immediately after extraction (1st row), overview of tumor and underlying CAM tissue (H&E staining, 2nd and 3rd rows), immunohistochemical staining of hnRNP K-expressing cells (4th row), Ki-67 + proliferative cells (5th row) and desmin + pericytes indicating angiogenesis (6th row). (C) Percentage of solid tumor formation of Cal-27 cells 4 days after xenotransplantation (9-10 tumors/group). (D) Percentage of proliferating Ki-67 + cells in Cal-27 xenografts. A total of 261-358 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 4 tumors/group. (E) Mean tumor volume of UPCI-SCC-154 cancer xenografts 4 days after xenotransplantation as assessed immediately after extraction. Tumor volume was calculated according to the formula: π/6 × length × width 2 . Mean of 9-10 tumors/group. (F) Percentage of proliferating Ki-67 + cells. A total of 298-632 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 5 tumors/group. * P<0.05 vs. control (mock 0 Gy) (Kruskal Wallis test, Dunnett's post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K; HNSCC, head and neck squamous cell carcinoma; CAM, chorioallantoic membrane; H&E, hematoxylin and eosin.
    Figure Legend Snippet: Knockdown of hnRNP K inhibits growth of HNSSC xenografts on the chick egg CAM in vivo . Cells (mock or siRNA, ± irradiation) were seeded on the CAM of fertilized chick eggs 7 days after the start of incubation (1.5×10 6 cells/egg in medium/Matrigel 1:1). After an incubation period of 4 days at 37°C, the tumors were collected, imaged, fixed and embedded in paraffin for immunohistochemical analysis. Sections (5 μ m) were stained for hnRNP K, proliferation marker Ki-67 and the angiogenesis marker desmin. Each group contained 9-10 tumor-bearing eggs. (A and B) Representative images of tumor xenografts immediately after extraction (1st row), overview of tumor and underlying CAM tissue (H&E staining, 2nd and 3rd rows), immunohistochemical staining of hnRNP K-expressing cells (4th row), Ki-67 + proliferative cells (5th row) and desmin + pericytes indicating angiogenesis (6th row). (C) Percentage of solid tumor formation of Cal-27 cells 4 days after xenotransplantation (9-10 tumors/group). (D) Percentage of proliferating Ki-67 + cells in Cal-27 xenografts. A total of 261-358 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 4 tumors/group. (E) Mean tumor volume of UPCI-SCC-154 cancer xenografts 4 days after xenotransplantation as assessed immediately after extraction. Tumor volume was calculated according to the formula: π/6 × length × width 2 . Mean of 9-10 tumors/group. (F) Percentage of proliferating Ki-67 + cells. A total of 298-632 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 5 tumors/group. * P<0.05 vs. control (mock 0 Gy) (Kruskal Wallis test, Dunnett's post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K; HNSCC, head and neck squamous cell carcinoma; CAM, chorioallantoic membrane; H&E, hematoxylin and eosin.

    Techniques Used: In Vivo, Irradiation, Incubation, Immunohistochemical staining, Staining, Marker, Expressing

    p akt1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p akt1
    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, <t>AKT1</t> and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.
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    1) Product Images from "SLC25A18 has prognostic value in colorectal cancer and represses Warburg effect and cell proliferation via Wnt signaling"

    Article Title: SLC25A18 has prognostic value in colorectal cancer and represses Warburg effect and cell proliferation via Wnt signaling

    Journal: American Journal of Cancer Research

    doi:

    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.
    Figure Legend Snippet: SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.

    Techniques Used: Expressing, Western Blot

    p akt1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p akt1
    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, <t>AKT1</t> and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.
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    1) Product Images from "SLC25A18 has prognostic value in colorectal cancer and represses Warburg effect and cell proliferation via Wnt signaling"

    Article Title: SLC25A18 has prognostic value in colorectal cancer and represses Warburg effect and cell proliferation via Wnt signaling

    Journal: American Journal of Cancer Research

    doi:

    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.
    Figure Legend Snippet: SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.

    Techniques Used: Expressing, Western Blot

    anti oct 4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti oct 4
    PL reduced the expression of the cancer stem cell markers SOX2, <t>Oct-4,</t> and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
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    1) Product Images from "Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer"

    Article Title: Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7486

    PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
    Figure Legend Snippet: PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.

    Techniques Used: Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot

    anti oct 4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti oct 4
    PL reduced the expression of the cancer stem cell markers SOX2, <t>Oct-4,</t> and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
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    1) Product Images from "Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer"

    Article Title: Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7486

    PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
    Figure Legend Snippet: PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.

    Techniques Used: Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot

    hnrnpk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc hnrnpk
    Hnrnpk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hnrnp k  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc hnrnp k
    ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and <t>hnRNP</t> <t>K</t> with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).
    Hnrnp K, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype"

    Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

    Journal: Nature Communications

    doi: 10.1038/ncomms10180

    ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and hnRNP K with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).
    Figure Legend Snippet: ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and hnRNP K with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Techniques Used: Derivative Assay, Sequencing, Western Blot, Transfection, Mutagenesis, Translocation Assay, Immunohistochemistry

    ( a ) Chromatin from MDA-MB-231 cells was extracted for ChIP and re-ChIP analysis. Results were normalized by input. ( b ) MDA-MB-231 cells were transfected with siRNA against negative control (NC), AURKA or hnRNP K for 48 h. ChIP assays were performed. ( c ) MDA-MB-231 cells overexpressing HA-tagged AURKA were transfected with hnRNP K or NC siRNA. After 24 h, cells were transfected with MYC promoter reporter (MYC) or basic reporter (Vec) along with pRL-TK for another 24 h. Dual-luciferase reporter assay was performed. ( d ) Mutated reporter along with pRL-TK were co-transfected with AURKA or/and hnRNP K plasmids for 24 h. Dual-luciferase reporter assay was performed. ( e ) 293T cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA for 48 h. S1 nuclease protection assay (SNPA) was performed to monitor MYC P1 and P2 transcripts. ( f ) Structure of chimeric gene consists of firefly luciferase and MYC promoter (−226/+211) (upper panel). Lower panel shows probes used for SNPA. ‘CT' represented CT element. The mutant chimeric gene was mutated at hnRNP K-binding site. ( g ) Chimeric gene or its mutant were co-transfected with AURKA and pRL-TK plasmids into 293T cells for 24 h. SNPA was performed using the probes shown in f . In parallel, a fraction of these cells was evaluated for Renilla luciferase activity, which reflects the transfection efficiency. ( h , i , j ) 293T cells were transfected with hnRNP K siRNA for 24 h. Cells were then co-transfected with WT or NLS-deleted hnRNP K, AURKA and MYC promoter or basic reporter along with pRL-TK for another 24 h. Transfected cells were harvested for immunoblotting (IB) ( h ), dual-luciferase reporter ( i ) and real-time PCR ( j ) analysis. ( k ) MCF-10A cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA. After 48 h, cells were collected to analyse CD24/CD44. ( l ) hnRNP K was knocked down in AURKA-overexpressing or control 10A-K-Ras (G12V) cells. Cells were sorted according to CD24 expression. CD24 Low population was used to perform limiting dilution assays. Bar represented the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).
    Figure Legend Snippet: ( a ) Chromatin from MDA-MB-231 cells was extracted for ChIP and re-ChIP analysis. Results were normalized by input. ( b ) MDA-MB-231 cells were transfected with siRNA against negative control (NC), AURKA or hnRNP K for 48 h. ChIP assays were performed. ( c ) MDA-MB-231 cells overexpressing HA-tagged AURKA were transfected with hnRNP K or NC siRNA. After 24 h, cells were transfected with MYC promoter reporter (MYC) or basic reporter (Vec) along with pRL-TK for another 24 h. Dual-luciferase reporter assay was performed. ( d ) Mutated reporter along with pRL-TK were co-transfected with AURKA or/and hnRNP K plasmids for 24 h. Dual-luciferase reporter assay was performed. ( e ) 293T cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA for 48 h. S1 nuclease protection assay (SNPA) was performed to monitor MYC P1 and P2 transcripts. ( f ) Structure of chimeric gene consists of firefly luciferase and MYC promoter (−226/+211) (upper panel). Lower panel shows probes used for SNPA. ‘CT' represented CT element. The mutant chimeric gene was mutated at hnRNP K-binding site. ( g ) Chimeric gene or its mutant were co-transfected with AURKA and pRL-TK plasmids into 293T cells for 24 h. SNPA was performed using the probes shown in f . In parallel, a fraction of these cells was evaluated for Renilla luciferase activity, which reflects the transfection efficiency. ( h , i , j ) 293T cells were transfected with hnRNP K siRNA for 24 h. Cells were then co-transfected with WT or NLS-deleted hnRNP K, AURKA and MYC promoter or basic reporter along with pRL-TK for another 24 h. Transfected cells were harvested for immunoblotting (IB) ( h ), dual-luciferase reporter ( i ) and real-time PCR ( j ) analysis. ( k ) MCF-10A cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA. After 48 h, cells were collected to analyse CD24/CD44. ( l ) hnRNP K was knocked down in AURKA-overexpressing or control 10A-K-Ras (G12V) cells. Cells were sorted according to CD24 expression. CD24 Low population was used to perform limiting dilution assays. Bar represented the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Techniques Used: Transfection, Negative Control, Luciferase, Reporter Assay, Mutagenesis, Binding Assay, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    ( a , b and c ) Primary breast cancer cells were isolated from breast cancer tissues derived from ten patients. Nuclear and cytoplasmic fractions were subjected to immunoblotting (IB) analysis ( a ). The expression of CD24 and CD44 were analysed using flow cytometry ( b ). Nuclear and cytoplasmic fractions of AURKA were normalized using histone H3 and β-actin, respectively. The nuclear fraction of c-Myc and hnRNP K were normalized using histone H3. The normalized AURKA, c-Myc, hnRNP K and the CD24 low /CD44 high population derived from the same patient were used to compose a scatterplot and linear regression analysis performed ( c ). ( d ) Breast cancer tissues were subjected to IHC staining for indicated antibodies. Representative images were acquired with × 10 and × 40 objectives (upper panel). Scale bar, 50 μm. Pearson's χ 2 -test was used to analyse the correlation between nuclear AURKA and c-Myc/CD24 (lower panel). ( e ) IHC staining for AURKA and c-Myc was performed on breast cancer tissues. Kaplan–Meier analysis was performed and log-rank test used to make statistical comparisons. ( f ) During breast cancer development, AURKA is overexpressed and translocates to the nucleus, where the nuclear AURKA acts as a transactivating factor that binds and activates the MYC promoter through its interaction with hnRNP K. Through these mechanisms, nuclear AURKA enhances BCSC phenotype. Importantly, these processes are dependent on the nuclear localization of AURKA rather than its kinase activity. Traditional targeted therapies focus on the inhibition of kinase activity, which may be insufficient for suppressing the kinase-independent oncogenic actions. These new findings suggest that targeting the spatial deregulation of kinase could be a promising strategy for overcoming kinase inhibitor insensitivity.
    Figure Legend Snippet: ( a , b and c ) Primary breast cancer cells were isolated from breast cancer tissues derived from ten patients. Nuclear and cytoplasmic fractions were subjected to immunoblotting (IB) analysis ( a ). The expression of CD24 and CD44 were analysed using flow cytometry ( b ). Nuclear and cytoplasmic fractions of AURKA were normalized using histone H3 and β-actin, respectively. The nuclear fraction of c-Myc and hnRNP K were normalized using histone H3. The normalized AURKA, c-Myc, hnRNP K and the CD24 low /CD44 high population derived from the same patient were used to compose a scatterplot and linear regression analysis performed ( c ). ( d ) Breast cancer tissues were subjected to IHC staining for indicated antibodies. Representative images were acquired with × 10 and × 40 objectives (upper panel). Scale bar, 50 μm. Pearson's χ 2 -test was used to analyse the correlation between nuclear AURKA and c-Myc/CD24 (lower panel). ( e ) IHC staining for AURKA and c-Myc was performed on breast cancer tissues. Kaplan–Meier analysis was performed and log-rank test used to make statistical comparisons. ( f ) During breast cancer development, AURKA is overexpressed and translocates to the nucleus, where the nuclear AURKA acts as a transactivating factor that binds and activates the MYC promoter through its interaction with hnRNP K. Through these mechanisms, nuclear AURKA enhances BCSC phenotype. Importantly, these processes are dependent on the nuclear localization of AURKA rather than its kinase activity. Traditional targeted therapies focus on the inhibition of kinase activity, which may be insufficient for suppressing the kinase-independent oncogenic actions. These new findings suggest that targeting the spatial deregulation of kinase could be a promising strategy for overcoming kinase inhibitor insensitivity.

    Techniques Used: Isolation, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Immunohistochemistry, Activity Assay, Inhibition

    hnrnp k rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc hnrnp k rabbit mab
    Hnrnp K Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti hnrnp k monoclonal antibody
    Expression patterns of <t>hnRNP</t> <t>K</t> in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.
    Anti Hnrnp K Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse monoclonal anti hnrnp k
    Association of nuclear and cytoplasmic <t> hnRNP K </t> expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.
    Mouse Monoclonal Anti Hnrnp K, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p akt1
    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, <t>AKT1</t> and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.
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    PL reduced the expression of the cancer stem cell markers SOX2, <t>Oct-4,</t> and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
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    Cell Signaling Technology Inc hnrnpk
    PL reduced the expression of the cancer stem cell markers SOX2, <t>Oct-4,</t> and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
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    Cell Signaling Technology Inc hnrnp k
    ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and <t>hnRNP</t> <t>K</t> with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).
    Hnrnp K, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc hnrnp k rabbit mab
    ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and <t>hnRNP</t> <t>K</t> with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).
    Hnrnp K Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.

    Journal: Oncology Letters

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    doi: 10.3892/ol.2020.12257

    Figure Lengend Snippet: Expression patterns of hnRNP K in HNSCC tissues and cells. (A) mRNA expression levels of hnRNP K in different cancerous and normal tissues from the Oncomine database. The color was determined by the best gene rank percentile for the analyses within the cell; red indicates upregulation, while blue indicates downregulation (fold-change ≥2). Numbers in each cell represent the number of studies reporting significant results. (B) Association between mRNA expression levels of hnRNP K and the overall survival of HNSCC according to data from the tumor-immune system interactions database. The survival curves were compared using the Kaplan Meier method according to the expression levels of hnRNP K. The log-rank test was performed to evaluate the statistical significance (P<0.05). (C) mRNA expression levels of hnRNP K in two HNSCC cell lines compared with WI-38 human embryonic lung fibroblasts were determined using reverse transcription-quantitative PCR. GAPDH served as the endogenous loading control. **P<0.01, ***P<0.001. (D) hnRNP K protein expression levels in HNSCC cell lines and WI-38 cells were analyzed using western blotting. β-actin was used as the loading control. (E) Immunohistochemical analysis of hnRNP K expression levels in 20 HNSCC and adjacent normal tissue samples (magnification, left ×10; right ×20). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogeneous nuclear ribonucleoprotein K.

    Article Snippet: Subsequently, the tissue sections were incubated with an anti-hnRNP K monoclonal antibody (1:250) overnight at 4°C, followed by incubation with anti-rabbit IgG, HRP-linked secondary antibody (1:5,000; cat. no. 7074) which was purchased from Cell Signaling Technology, Inc., for 1 h at room temperature.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining

    hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.

    Journal: Oncology Letters

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    doi: 10.3892/ol.2020.12257

    Figure Lengend Snippet: hnRNP K knockdown inhibits CAL-27 cell viability, proliferation and migration. The transfection efficiency of hnRNP K knockdown using shRNA was analyzed using (A) western blotting and (B) reverse transcription-quantitative PCR. CAL-27 cell viability was determining using a (C) Cell Counting Kit-8 assay and (D) an absolute count assay following the knockdown of hnRNP K. (E) Representative images of the colony formation assay used to evaluate the proliferation of CAL-27 cells after knocking down the expression of hnRNP K. (F) Semi-quantification of the results of the colony formation assay presented in part (E). (G) An EdU incorporation assay was used to indicate the percentage of proliferated CAL-27 cells following hnRNP K knockdown. (H) Wound healing assay was used to determine the migratory ability of CAL-27 cells following the knockdown of hnRNP K (magnification, ×10). (I) Semi-quantification of the wound healing assay results form part (H). (J) hnRNP K knockdown decreased the cell migration of CAL-27 cells, as determined using a Transwell assay. Magnification, ×10. (K) Semi-quantification of the number of migratory cells from part (J). Error bars represent the mean ± SD of three independent experiments, except for in part (K), where they represent the mean ± SD of five randomly selected fields of view. *P<0.05, **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh/shRNA, short hairpin RNA; NC, negative control; OD, optical density; EdU, 5-Ethynyl-2′-deoxyuridine.

    Article Snippet: Subsequently, the tissue sections were incubated with an anti-hnRNP K monoclonal antibody (1:250) overnight at 4°C, followed by incubation with anti-rabbit IgG, HRP-linked secondary antibody (1:5,000; cat. no. 7074) which was purchased from Cell Signaling Technology, Inc., for 1 h at room temperature.

    Techniques: Migration, Transfection, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Colony Assay, Expressing, Wound Healing Assay, Transwell Assay, Negative Control

    hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.

    Journal: Oncology Letters

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    doi: 10.3892/ol.2020.12257

    Figure Lengend Snippet: hnRNP K knockdown suppresses the growth of CAL-27 ×enograft tumors in vivo . (A) Size of tumors derived from shhnRNP K- or shNC-transfected CAL-27 cells in six male nude mice/group are presented. (B) Tumor weight of the xenografts from the two groups. (C) Tumor volume of xenografts derived from the two groups were measured every 2 days (except the first 3 days) from the 5th day after injection to the study endpoint (at 4 weeks) and are presented as growth curves. (D) Body weight of the mice in the two groups at the indicated time-points. Error bars represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. shNC. hnRNP K, heterogeneous nuclear ribonucleoprotein K; sh, short hairpin RNA; NC, negative control.

    Article Snippet: Subsequently, the tissue sections were incubated with an anti-hnRNP K monoclonal antibody (1:250) overnight at 4°C, followed by incubation with anti-rabbit IgG, HRP-linked secondary antibody (1:5,000; cat. no. 7074) which was purchased from Cell Signaling Technology, Inc., for 1 h at room temperature.

    Techniques: In Vivo, Derivative Assay, Transfection, Injection, shRNA, Negative Control

    GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.

    Journal: Oncology Letters

    Article Title: Involvement of the Wnt/β-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma

    doi: 10.3892/ol.2020.12257

    Figure Lengend Snippet: GO functional term and KEGG signaling pathway enrichment analyses of hnRNP K-associated genes. (A) GO annotation of downregulated mRNAs with the top 10 enrichment scores in the categories of biological process, cellular components and molecular functions. (B) KEGG signaling pathway enrichment analysis of downregulated mRNAs with the top 30 enrichment scores. mRNA and protein expression levels of hnRNP K, β-Catenin, Dvl2, c-Jun, Met, Cyclin-D1, c-Myc and MMP7 were analyzed by (C) reverse transcription-quantitative PCR and (D) western blotting, respectively, following the knockdown of hnRNP K with siRNA in CAL-27 cells. (E) Semi-quantification of the expression levels presented in part (D). *P<0.05, **P<0.01, ***P<0.001 vs. shNC. (F) Transfection efficiency of β-Catenin overexpression plasmid was analyzed using western blotting. (G) Western blotting analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. (H) RT-qPCR analysis of flag, β-catenin, hnRNP K, c-Jun and c-Myc following the inhibition of hnRNP K using siRNA and the plasmid-mediated overexpression of β-Catenin in CAL-27 cells. *P<0.05, **P<0.01, ***P<0.001. GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes; hnRNP K, heterogeneous nuclear ribonucleoprotein K; Dvl2, disheveled 2; MMP7, matrix metalloproteinase 7; si/siRNA, small interfering RNA; NC, negative control.

    Article Snippet: Subsequently, the tissue sections were incubated with an anti-hnRNP K monoclonal antibody (1:250) overnight at 4°C, followed by incubation with anti-rabbit IgG, HRP-linked secondary antibody (1:5,000; cat. no. 7074) which was purchased from Cell Signaling Technology, Inc., for 1 h at room temperature.

    Techniques: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Plasmid Preparation, Inhibition, Quantitative RT-PCR, Small Interfering RNA, Negative Control

    Association of nuclear and cytoplasmic  hnRNP K  expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: Association of nuclear and cytoplasmic hnRNP K expression levels (IHC score) in HNSCC tissues with the clinicopathological characteristics of 117 patients with HNSCC.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques: Expressing

    (A) Representative images of HNSCC tissue samples for hnRNP K IHC staining focusing on nuclear (nuc) and cytoplasmic (cyt) hnRNP K expression (scale bar, 200 μ m): hnRNP K-negative (upper panel), nuclear hnRNP K expression (middle panel) and hnRNP K nuclear and cytoplasmic expression (lower panel). (B) Representative images of p16 INK4A-negative (upper panel) and -positive (lower panel) HNSCC samples. Staining for p16INK4A served as a surrogate marker for HPV-positive HNSCC. HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; IHC, immunohistochemistry; HPV, human papillomavirus.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: (A) Representative images of HNSCC tissue samples for hnRNP K IHC staining focusing on nuclear (nuc) and cytoplasmic (cyt) hnRNP K expression (scale bar, 200 μ m): hnRNP K-negative (upper panel), nuclear hnRNP K expression (middle panel) and hnRNP K nuclear and cytoplasmic expression (lower panel). (B) Representative images of p16 INK4A-negative (upper panel) and -positive (lower panel) HNSCC samples. Staining for p16INK4A served as a surrogate marker for HPV-positive HNSCC. HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; IHC, immunohistochemistry; HPV, human papillomavirus.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques: Immunohistochemistry, Expressing, Staining, Marker

    Association of  hnRNP K  IHC score with the analyzed categories (tissue, sex and stage) among 117 HNSCC cases and 15 normal oral tissue samples.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: Association of hnRNP K IHC score with the analyzed categories (tissue, sex and stage) among 117 HNSCC cases and 15 normal oral tissue samples.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques:

    (A) Representative images of HNSCC cell clusters stained for p16INK4a using immunofluorescence illustrate the HPV status of Cal-27 (HPV-negative) and UPCI-SCC 154 (HPV-positive) cells (scale bar, 40 μ m). (B) Clonogenic survival assay of the HNSCC cell lines Cal-27 and UPCI-SCC 154 after 9 days. Data are presented as mean ± SD of 4 independent experiments. (C) Representative immunoblots demonstrate a rapid increase in cellular hnRNP K levels induced by IR (2 Gy), reaching maximum levels after 30-60 min before normalization of cellular hnRNP K levels within 24 h. Ratios represent hnRNP K/GAPDH referenced to non-irradiated control. (D) Dose-dependent accumulation of cellular hnRNP K 1 h after IR. (E) Immunofluorescence microscopy indicated cytoplasmic hnRNP K accumulation 1 h after irradiating cells with 2 Gy (scale bar, 20 μ m). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; HPV, human papillomavirus; IR, ionizing radiation.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: (A) Representative images of HNSCC cell clusters stained for p16INK4a using immunofluorescence illustrate the HPV status of Cal-27 (HPV-negative) and UPCI-SCC 154 (HPV-positive) cells (scale bar, 40 μ m). (B) Clonogenic survival assay of the HNSCC cell lines Cal-27 and UPCI-SCC 154 after 9 days. Data are presented as mean ± SD of 4 independent experiments. (C) Representative immunoblots demonstrate a rapid increase in cellular hnRNP K levels induced by IR (2 Gy), reaching maximum levels after 30-60 min before normalization of cellular hnRNP K levels within 24 h. Ratios represent hnRNP K/GAPDH referenced to non-irradiated control. (D) Dose-dependent accumulation of cellular hnRNP K 1 h after IR. (E) Immunofluorescence microscopy indicated cytoplasmic hnRNP K accumulation 1 h after irradiating cells with 2 Gy (scale bar, 20 μ m). HNSCC, head and neck squamous cell carcinoma; hnRNP K, heterogenous nuclear ribonucleoprotein K; HPV, human papillomavirus; IR, ionizing radiation.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques: Staining, Immunofluorescence, Clonogenic Cell Survival Assay, Western Blot, Irradiation, Microscopy

    (A) Effective hnRNP K knockdown by transient transfection was verified by immunoblotting. Mock transfection served as control. (B) Representative images of colonies after 9 days of incubation. (C) Statistical analysis of clonogenic survival assays during hnRNP K knockdown. All experiments were carried out in quadruplicate. Data are presented as mean ± SD. * P<0.05 (Kruskal Wallis test, Tukey's post hoc test). (D) ELISA showed significantly increased levels of cellular active caspase-3 in Cal-27 and UPCI-SCC-154 cells parallel to hnRNP K knockdown. Data are presented as mean ± SD. * P<0.05; n.s., not significant; n=6 (Kruskal Wallis test, Holm-Sidak post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: (A) Effective hnRNP K knockdown by transient transfection was verified by immunoblotting. Mock transfection served as control. (B) Representative images of colonies after 9 days of incubation. (C) Statistical analysis of clonogenic survival assays during hnRNP K knockdown. All experiments were carried out in quadruplicate. Data are presented as mean ± SD. * P<0.05 (Kruskal Wallis test, Tukey's post hoc test). (D) ELISA showed significantly increased levels of cellular active caspase-3 in Cal-27 and UPCI-SCC-154 cells parallel to hnRNP K knockdown. Data are presented as mean ± SD. * P<0.05; n.s., not significant; n=6 (Kruskal Wallis test, Holm-Sidak post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques: Transfection, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

    Knockdown of hnRNP K inhibits growth of HNSSC xenografts on the chick egg CAM in vivo . Cells (mock or siRNA, ± irradiation) were seeded on the CAM of fertilized chick eggs 7 days after the start of incubation (1.5×10 6 cells/egg in medium/Matrigel 1:1). After an incubation period of 4 days at 37°C, the tumors were collected, imaged, fixed and embedded in paraffin for immunohistochemical analysis. Sections (5 μ m) were stained for hnRNP K, proliferation marker Ki-67 and the angiogenesis marker desmin. Each group contained 9-10 tumor-bearing eggs. (A and B) Representative images of tumor xenografts immediately after extraction (1st row), overview of tumor and underlying CAM tissue (H&E staining, 2nd and 3rd rows), immunohistochemical staining of hnRNP K-expressing cells (4th row), Ki-67 + proliferative cells (5th row) and desmin + pericytes indicating angiogenesis (6th row). (C) Percentage of solid tumor formation of Cal-27 cells 4 days after xenotransplantation (9-10 tumors/group). (D) Percentage of proliferating Ki-67 + cells in Cal-27 xenografts. A total of 261-358 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 4 tumors/group. (E) Mean tumor volume of UPCI-SCC-154 cancer xenografts 4 days after xenotransplantation as assessed immediately after extraction. Tumor volume was calculated according to the formula: π/6 × length × width 2 . Mean of 9-10 tumors/group. (F) Percentage of proliferating Ki-67 + cells. A total of 298-632 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 5 tumors/group. * P<0.05 vs. control (mock 0 Gy) (Kruskal Wallis test, Dunnett's post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K; HNSCC, head and neck squamous cell carcinoma; CAM, chorioallantoic membrane; H&E, hematoxylin and eosin.

    Journal: International Journal of Molecular Medicine

    Article Title: Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma

    doi: 10.3892/ijmm.2020.4718

    Figure Lengend Snippet: Knockdown of hnRNP K inhibits growth of HNSSC xenografts on the chick egg CAM in vivo . Cells (mock or siRNA, ± irradiation) were seeded on the CAM of fertilized chick eggs 7 days after the start of incubation (1.5×10 6 cells/egg in medium/Matrigel 1:1). After an incubation period of 4 days at 37°C, the tumors were collected, imaged, fixed and embedded in paraffin for immunohistochemical analysis. Sections (5 μ m) were stained for hnRNP K, proliferation marker Ki-67 and the angiogenesis marker desmin. Each group contained 9-10 tumor-bearing eggs. (A and B) Representative images of tumor xenografts immediately after extraction (1st row), overview of tumor and underlying CAM tissue (H&E staining, 2nd and 3rd rows), immunohistochemical staining of hnRNP K-expressing cells (4th row), Ki-67 + proliferative cells (5th row) and desmin + pericytes indicating angiogenesis (6th row). (C) Percentage of solid tumor formation of Cal-27 cells 4 days after xenotransplantation (9-10 tumors/group). (D) Percentage of proliferating Ki-67 + cells in Cal-27 xenografts. A total of 261-358 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 4 tumors/group. (E) Mean tumor volume of UPCI-SCC-154 cancer xenografts 4 days after xenotransplantation as assessed immediately after extraction. Tumor volume was calculated according to the formula: π/6 × length × width 2 . Mean of 9-10 tumors/group. (F) Percentage of proliferating Ki-67 + cells. A total of 298-632 cells from each tumor were evaluated. Data are presented as the mean ± SEM of 5 tumors/group. * P<0.05 vs. control (mock 0 Gy) (Kruskal Wallis test, Dunnett's post hoc test). hnRNP K, heterogenous nuclear ribonucleoprotein K; HNSCC, head and neck squamous cell carcinoma; CAM, chorioallantoic membrane; H&E, hematoxylin and eosin.

    Article Snippet: For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT.

    Techniques: In Vivo, Irradiation, Incubation, Immunohistochemical staining, Staining, Marker, Expressing

    SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.

    Journal: American Journal of Cancer Research

    Article Title: SLC25A18 has prognostic value in colorectal cancer and represses Warburg effect and cell proliferation via Wnt signaling

    doi:

    Figure Lengend Snippet: SLC25A18 negatively regulates genes expression related to aerobics glycolysis, cell proliferation, and Wnt/β-catenin cascade. (A) Western blotting verified a negative protein level of genes such as MYC, PKM2, LDHA, AKT1 and pAKT1 which are involved in cell aerobics glycolysis and cell proliferation when SLC25A18 was overexpressed or knocked down. (B) Gene set enrichment analysis implied a negative relationship between highly expressed SLC25A18 and the enrichment of CTNNB1 target genes and Wnt/β-catenin Signaling (HALLMARK_ WNT_BETA_CATENIN_SIGNALING, REACTOME_SIGNALING_BY_WNT) in patients with CRC from the TCGA database, (C) which was also verified by western blotting. All experiments were conducted in triplicates.

    Article Snippet: Target proteins, include SLC25A18 (Proteintech, Wuhan, China, 17348-1-AP), β-catenin (Cell Signaling Technology, Danvers, MA, #9587), c-Myc (Abcam, Cambridge, MA, Ab32072), PKM2 (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab137852","term_id":"62158433","term_text":"AB137852"}} Ab137852 ), LHDA (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab125683","term_id":"47076356","term_text":"AB125683"}} Ab125683 ), TCF4 (Affinity, DF6275), TCF1 (Affinity, DF7180), AKT1 (Cell Signaling Technology, Danvers, MA#2938), p-AKT1 (Cell Signaling Technology, #9081), and GAPDH (Cell Signaling Technology, #5174).

    Techniques: Expressing, Western Blot

    PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.

    Journal: Oncology Letters

    Article Title: Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer

    doi: 10.3892/ol.2017.7486

    Figure Lengend Snippet: PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.

    Article Snippet: The following primary antibodies were used in the present study: Anti-E-cadherin (cat. no. 24E10, dilution, 1:1,000), anti-N-cadherin (cat. no. D4R1HN, dilution, 1:1,000), anti-vimentin (cat. no. D21H3, dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Snail (cat. no. SC-28199, dilution, 1:1,000), anti-Slug (cat. no. SC-10436, dilution, 1:1,000), anti-Oct-4 (cat. no. SC-9081, dilution, 1:1,000), and anti-CK18 (cat. no. SC-6259, dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-NANOG (cat. no. ab109250, dilution, 1:1,000; Abcam, Cambridge, UK), anti-SOX2 (cat. no. AB5603, dilution, 1:1,000; EMD Millipore, Billerica, MA, USA), and anti-GAPDH (cat. no. GTX100118, dilution, 1:8,000; GeneTex, Inc., Irvine, CA, USA).

    Techniques: Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot

    ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and hnRNP K with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Journal: Nature Communications

    Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

    doi: 10.1038/ncomms10180

    Figure Lengend Snippet: ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and hnRNP K with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Article Snippet: The chromatin (25 μg) was immunoprecipitated for 12 h with 2 μg of specific antibodies against AURKA (Millipore), hnRNP K (Cell Signaling) or IgG (rabbit, Santa Cruz) and Protein G magnetic beads (25 μl).

    Techniques: Derivative Assay, Sequencing, Western Blot, Transfection, Mutagenesis, Translocation Assay, Immunohistochemistry

    ( a ) Chromatin from MDA-MB-231 cells was extracted for ChIP and re-ChIP analysis. Results were normalized by input. ( b ) MDA-MB-231 cells were transfected with siRNA against negative control (NC), AURKA or hnRNP K for 48 h. ChIP assays were performed. ( c ) MDA-MB-231 cells overexpressing HA-tagged AURKA were transfected with hnRNP K or NC siRNA. After 24 h, cells were transfected with MYC promoter reporter (MYC) or basic reporter (Vec) along with pRL-TK for another 24 h. Dual-luciferase reporter assay was performed. ( d ) Mutated reporter along with pRL-TK were co-transfected with AURKA or/and hnRNP K plasmids for 24 h. Dual-luciferase reporter assay was performed. ( e ) 293T cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA for 48 h. S1 nuclease protection assay (SNPA) was performed to monitor MYC P1 and P2 transcripts. ( f ) Structure of chimeric gene consists of firefly luciferase and MYC promoter (−226/+211) (upper panel). Lower panel shows probes used for SNPA. ‘CT' represented CT element. The mutant chimeric gene was mutated at hnRNP K-binding site. ( g ) Chimeric gene or its mutant were co-transfected with AURKA and pRL-TK plasmids into 293T cells for 24 h. SNPA was performed using the probes shown in f . In parallel, a fraction of these cells was evaluated for Renilla luciferase activity, which reflects the transfection efficiency. ( h , i , j ) 293T cells were transfected with hnRNP K siRNA for 24 h. Cells were then co-transfected with WT or NLS-deleted hnRNP K, AURKA and MYC promoter or basic reporter along with pRL-TK for another 24 h. Transfected cells were harvested for immunoblotting (IB) ( h ), dual-luciferase reporter ( i ) and real-time PCR ( j ) analysis. ( k ) MCF-10A cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA. After 48 h, cells were collected to analyse CD24/CD44. ( l ) hnRNP K was knocked down in AURKA-overexpressing or control 10A-K-Ras (G12V) cells. Cells were sorted according to CD24 expression. CD24 Low population was used to perform limiting dilution assays. Bar represented the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Journal: Nature Communications

    Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

    doi: 10.1038/ncomms10180

    Figure Lengend Snippet: ( a ) Chromatin from MDA-MB-231 cells was extracted for ChIP and re-ChIP analysis. Results were normalized by input. ( b ) MDA-MB-231 cells were transfected with siRNA against negative control (NC), AURKA or hnRNP K for 48 h. ChIP assays were performed. ( c ) MDA-MB-231 cells overexpressing HA-tagged AURKA were transfected with hnRNP K or NC siRNA. After 24 h, cells were transfected with MYC promoter reporter (MYC) or basic reporter (Vec) along with pRL-TK for another 24 h. Dual-luciferase reporter assay was performed. ( d ) Mutated reporter along with pRL-TK were co-transfected with AURKA or/and hnRNP K plasmids for 24 h. Dual-luciferase reporter assay was performed. ( e ) 293T cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA for 48 h. S1 nuclease protection assay (SNPA) was performed to monitor MYC P1 and P2 transcripts. ( f ) Structure of chimeric gene consists of firefly luciferase and MYC promoter (−226/+211) (upper panel). Lower panel shows probes used for SNPA. ‘CT' represented CT element. The mutant chimeric gene was mutated at hnRNP K-binding site. ( g ) Chimeric gene or its mutant were co-transfected with AURKA and pRL-TK plasmids into 293T cells for 24 h. SNPA was performed using the probes shown in f . In parallel, a fraction of these cells was evaluated for Renilla luciferase activity, which reflects the transfection efficiency. ( h , i , j ) 293T cells were transfected with hnRNP K siRNA for 24 h. Cells were then co-transfected with WT or NLS-deleted hnRNP K, AURKA and MYC promoter or basic reporter along with pRL-TK for another 24 h. Transfected cells were harvested for immunoblotting (IB) ( h ), dual-luciferase reporter ( i ) and real-time PCR ( j ) analysis. ( k ) MCF-10A cells overexpressing HA-AURKA were transfected with hnRNP K or NC siRNA. After 48 h, cells were collected to analyse CD24/CD44. ( l ) hnRNP K was knocked down in AURKA-overexpressing or control 10A-K-Ras (G12V) cells. Cells were sorted according to CD24 expression. CD24 Low population was used to perform limiting dilution assays. Bar represented the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

    Article Snippet: The chromatin (25 μg) was immunoprecipitated for 12 h with 2 μg of specific antibodies against AURKA (Millipore), hnRNP K (Cell Signaling) or IgG (rabbit, Santa Cruz) and Protein G magnetic beads (25 μl).

    Techniques: Transfection, Negative Control, Luciferase, Reporter Assay, Mutagenesis, Binding Assay, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    ( a , b and c ) Primary breast cancer cells were isolated from breast cancer tissues derived from ten patients. Nuclear and cytoplasmic fractions were subjected to immunoblotting (IB) analysis ( a ). The expression of CD24 and CD44 were analysed using flow cytometry ( b ). Nuclear and cytoplasmic fractions of AURKA were normalized using histone H3 and β-actin, respectively. The nuclear fraction of c-Myc and hnRNP K were normalized using histone H3. The normalized AURKA, c-Myc, hnRNP K and the CD24 low /CD44 high population derived from the same patient were used to compose a scatterplot and linear regression analysis performed ( c ). ( d ) Breast cancer tissues were subjected to IHC staining for indicated antibodies. Representative images were acquired with × 10 and × 40 objectives (upper panel). Scale bar, 50 μm. Pearson's χ 2 -test was used to analyse the correlation between nuclear AURKA and c-Myc/CD24 (lower panel). ( e ) IHC staining for AURKA and c-Myc was performed on breast cancer tissues. Kaplan–Meier analysis was performed and log-rank test used to make statistical comparisons. ( f ) During breast cancer development, AURKA is overexpressed and translocates to the nucleus, where the nuclear AURKA acts as a transactivating factor that binds and activates the MYC promoter through its interaction with hnRNP K. Through these mechanisms, nuclear AURKA enhances BCSC phenotype. Importantly, these processes are dependent on the nuclear localization of AURKA rather than its kinase activity. Traditional targeted therapies focus on the inhibition of kinase activity, which may be insufficient for suppressing the kinase-independent oncogenic actions. These new findings suggest that targeting the spatial deregulation of kinase could be a promising strategy for overcoming kinase inhibitor insensitivity.

    Journal: Nature Communications

    Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

    doi: 10.1038/ncomms10180

    Figure Lengend Snippet: ( a , b and c ) Primary breast cancer cells were isolated from breast cancer tissues derived from ten patients. Nuclear and cytoplasmic fractions were subjected to immunoblotting (IB) analysis ( a ). The expression of CD24 and CD44 were analysed using flow cytometry ( b ). Nuclear and cytoplasmic fractions of AURKA were normalized using histone H3 and β-actin, respectively. The nuclear fraction of c-Myc and hnRNP K were normalized using histone H3. The normalized AURKA, c-Myc, hnRNP K and the CD24 low /CD44 high population derived from the same patient were used to compose a scatterplot and linear regression analysis performed ( c ). ( d ) Breast cancer tissues were subjected to IHC staining for indicated antibodies. Representative images were acquired with × 10 and × 40 objectives (upper panel). Scale bar, 50 μm. Pearson's χ 2 -test was used to analyse the correlation between nuclear AURKA and c-Myc/CD24 (lower panel). ( e ) IHC staining for AURKA and c-Myc was performed on breast cancer tissues. Kaplan–Meier analysis was performed and log-rank test used to make statistical comparisons. ( f ) During breast cancer development, AURKA is overexpressed and translocates to the nucleus, where the nuclear AURKA acts as a transactivating factor that binds and activates the MYC promoter through its interaction with hnRNP K. Through these mechanisms, nuclear AURKA enhances BCSC phenotype. Importantly, these processes are dependent on the nuclear localization of AURKA rather than its kinase activity. Traditional targeted therapies focus on the inhibition of kinase activity, which may be insufficient for suppressing the kinase-independent oncogenic actions. These new findings suggest that targeting the spatial deregulation of kinase could be a promising strategy for overcoming kinase inhibitor insensitivity.

    Article Snippet: The chromatin (25 μg) was immunoprecipitated for 12 h with 2 μg of specific antibodies against AURKA (Millipore), hnRNP K (Cell Signaling) or IgG (rabbit, Santa Cruz) and Protein G magnetic beads (25 μl).

    Techniques: Isolation, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Immunohistochemistry, Activity Assay, Inhibition