ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ulk1
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ulk1/product/Cell Signaling Technology Inc
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    ulk1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc ulk1
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ulk1/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
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    anti ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ulk1
    Correlation between <t>ULK1/2,</t> YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.
    Anti Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ulk1/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ulk1 - by Bioz Stars, 2023-02
    98/100 stars

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    1) Product Images from "Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development"

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.60018

    Correlation between ULK1/2, YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.
    Figure Legend Snippet: Correlation between ULK1/2, YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.

    Techniques Used: Immunohistochemistry, Expressing, Staining, Western Blot

    Kinase activity of ULK1/2 is instrumental for hypoxic glycolysis mediated by PKM2 independent of BNIP3. (A) Western-blotting comparing the levels of ULK1 and ULK2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia, respectively. GAPDH was used as internal control of cytoplasmic lysates. Scr, scrambled shRNA; WB, western-blotting. (B and C) Glucose consumption (B) or lactate production (C) of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D and E) Glucose consumption (D) or lactate production (E) of SW-1990 cells treated with SBI-0206965 (SBI) at the indicated concentrations under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus D. D, DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Western-blotting detecting the amount of ULK1, ULK2 and BNIP3 expression in SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (G and H) Glucose consumption (G) or lactate production (H) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) Western-blotting assessing the amount of BNIP3 expression in SW-1990 cells with BNIP3 shRNA (sh.BNIP3) transfection. (J and K) Glucose consumption (J) or lactate production (K) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided Student's t test was used to calculate the P value. (L) Western-blotting detecting the amount of ULK1, ULK2 and PKM2 expression in SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (M and N) Glucose consumption (M) or lactate production (N) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. N.S., no significant. (O) Western-blotting examining the amount of PKM2 expression in SW-1990 cells with PKM2 shRNA (sh.PKM2) transfection. (P and Q) Glucose consumption (P) or lactate production (Q) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided Student's t test was used to calculate the P value.
    Figure Legend Snippet: Kinase activity of ULK1/2 is instrumental for hypoxic glycolysis mediated by PKM2 independent of BNIP3. (A) Western-blotting comparing the levels of ULK1 and ULK2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia, respectively. GAPDH was used as internal control of cytoplasmic lysates. Scr, scrambled shRNA; WB, western-blotting. (B and C) Glucose consumption (B) or lactate production (C) of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D and E) Glucose consumption (D) or lactate production (E) of SW-1990 cells treated with SBI-0206965 (SBI) at the indicated concentrations under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus D. D, DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Western-blotting detecting the amount of ULK1, ULK2 and BNIP3 expression in SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (G and H) Glucose consumption (G) or lactate production (H) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) Western-blotting assessing the amount of BNIP3 expression in SW-1990 cells with BNIP3 shRNA (sh.BNIP3) transfection. (J and K) Glucose consumption (J) or lactate production (K) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided Student's t test was used to calculate the P value. (L) Western-blotting detecting the amount of ULK1, ULK2 and PKM2 expression in SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (M and N) Glucose consumption (M) or lactate production (N) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. N.S., no significant. (O) Western-blotting examining the amount of PKM2 expression in SW-1990 cells with PKM2 shRNA (sh.PKM2) transfection. (P and Q) Glucose consumption (P) or lactate production (Q) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided Student's t test was used to calculate the P value.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, shRNA

    YAP transactivates PKM2 in an ULK1/2-dependent fashion under hypoxia. (A) Western-blotting determining the amount of PKM2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Scr, scrambled shRNA. (B) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C) Representative IHC staining images (c1) and quantification (c2) for PKM2 staining in the xenografts from mice injected with SW-1990 cells expressing ULK1, ULK2 or ULK1/2 shRNA ( n =6). Scale bar = 50 μm. (D) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ChIP analysis for YAP and HIF-1α binding to PKM2 gene promoter in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of hypoxia, respectively. (F) Coimmunoprecipitation assay testing the interaction between nuclear YAP and HIF-1α in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. (G) RT-qPCR analyses of YAP1 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 and *** P <0.001 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Western-blotting comparing the abundance of PKM2 expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia. (I) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value. (J) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value.
    Figure Legend Snippet: YAP transactivates PKM2 in an ULK1/2-dependent fashion under hypoxia. (A) Western-blotting determining the amount of PKM2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Scr, scrambled shRNA. (B) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C) Representative IHC staining images (c1) and quantification (c2) for PKM2 staining in the xenografts from mice injected with SW-1990 cells expressing ULK1, ULK2 or ULK1/2 shRNA ( n =6). Scale bar = 50 μm. (D) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ChIP analysis for YAP and HIF-1α binding to PKM2 gene promoter in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of hypoxia, respectively. (F) Coimmunoprecipitation assay testing the interaction between nuclear YAP and HIF-1α in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. (G) RT-qPCR analyses of YAP1 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 and *** P <0.001 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Western-blotting comparing the abundance of PKM2 expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia. (I) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value. (J) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value.

    Techniques Used: Western Blot, Expressing, Transfection, shRNA, Quantitative RT-PCR, Immunohistochemistry, Staining, Injection, Luciferase, Activity Assay, Binding Assay, Co-Immunoprecipitation Assay, Knock-Out

    ULK1 translocates into nucleus and phosphorylates YAP at Ser227 during hypoxia, which leads to YAP stabilization. (A) CHX pulse-chase experiments comparing the turnover of YAP protein in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of 20 μg/mL CHX treatment for the indicated times under hypoxia, respectively. (B) Western blotting analyses testing abundance of YAP protein in the hypoxia-stimulated SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of 10 μmol/L MG132 treatment, respectively. (C) Cellular ubiquitination assays examining the poly-Ub levels of YAP in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. PD, pull-down; Ni-NTA, Ni 2+ -nitrilotriacetic acid (NTA). (D) Coimmunoprecipitation assay evaluating the interaction between ULK1 and nuclear YAP in SW-1990 cells with or without hypoxia stimuli. (E) His-pulldown assay for determination of ULK1-YAP interaction with mixing purified His-tagged ULK1 immobilized on Ni 2+ -nitrilotriacetic acid (NTA)-sepharose beads and nuclear lysates from hypoxia-stimulated SW-1990 cells expressing Flag-tagged wild-type YAP followed by WB analyses of proteins on beads with an anti-Flag antibody. PD, pull-down. (F) Sequence alignment of the evolutionarily conserved ULK1-phosphorylating motif 224 MMN*SA 228 peptide in amino acid sequence of YAP protein among human and other primates. (G) Left panel: western-blotting examining the levels of ULK1 protein in the SW-1990 cells transfected with ULK1 shRNA. Right panel: coimmunoprecipitation assay detecting the abundance of Flag-tagged wild-type YAP phosphorylation in hypoxia-stimulated SW-1990 cells in the presence or absence of ULK1 shRNA transfection with an anti-phospho-serine antibody. (H) Coimmunoprecipitation assay comparing the levels of Flag-tagged wild-type YAP (WT), mutant YAP Ser227A (S227A) and Ser227E (S227E) phosphorylation in hypoxia-stimulated SW-1990 cells with an anti-phospho-serine antibody. (I) In vitro kinase assay with mixing His-tagged ULK1 protein and IPs of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) followed by WB analyses with an anti-phospho-serine antibody. (J) Schematic defining the role of ULK1/2 in Ser227 phosphorylation and stabilization of nuclear YAP during PDAC development in response to hypoxia sensing.
    Figure Legend Snippet: ULK1 translocates into nucleus and phosphorylates YAP at Ser227 during hypoxia, which leads to YAP stabilization. (A) CHX pulse-chase experiments comparing the turnover of YAP protein in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of 20 μg/mL CHX treatment for the indicated times under hypoxia, respectively. (B) Western blotting analyses testing abundance of YAP protein in the hypoxia-stimulated SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of 10 μmol/L MG132 treatment, respectively. (C) Cellular ubiquitination assays examining the poly-Ub levels of YAP in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. PD, pull-down; Ni-NTA, Ni 2+ -nitrilotriacetic acid (NTA). (D) Coimmunoprecipitation assay evaluating the interaction between ULK1 and nuclear YAP in SW-1990 cells with or without hypoxia stimuli. (E) His-pulldown assay for determination of ULK1-YAP interaction with mixing purified His-tagged ULK1 immobilized on Ni 2+ -nitrilotriacetic acid (NTA)-sepharose beads and nuclear lysates from hypoxia-stimulated SW-1990 cells expressing Flag-tagged wild-type YAP followed by WB analyses of proteins on beads with an anti-Flag antibody. PD, pull-down. (F) Sequence alignment of the evolutionarily conserved ULK1-phosphorylating motif 224 MMN*SA 228 peptide in amino acid sequence of YAP protein among human and other primates. (G) Left panel: western-blotting examining the levels of ULK1 protein in the SW-1990 cells transfected with ULK1 shRNA. Right panel: coimmunoprecipitation assay detecting the abundance of Flag-tagged wild-type YAP phosphorylation in hypoxia-stimulated SW-1990 cells in the presence or absence of ULK1 shRNA transfection with an anti-phospho-serine antibody. (H) Coimmunoprecipitation assay comparing the levels of Flag-tagged wild-type YAP (WT), mutant YAP Ser227A (S227A) and Ser227E (S227E) phosphorylation in hypoxia-stimulated SW-1990 cells with an anti-phospho-serine antibody. (I) In vitro kinase assay with mixing His-tagged ULK1 protein and IPs of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) followed by WB analyses with an anti-phospho-serine antibody. (J) Schematic defining the role of ULK1/2 in Ser227 phosphorylation and stabilization of nuclear YAP during PDAC development in response to hypoxia sensing.

    Techniques Used: Pulse Chase, Transfection, shRNA, Western Blot, Co-Immunoprecipitation Assay, Purification, Expressing, Sequencing, Mutagenesis, In Vitro, Kinase Assay

    Ser227 phosphorylation stabilizes YAP and augments PKM2 transcription upon hypoxia. (A) Representative immunfluorescence images of ULK1 nuclear localization in SW-1990 cells before and after hypoxia stimuli. Scale bar = 20 μm. (B) Representative immunfluorescence images detecting the localization of ULK1 and YAP in YAP knockout (KO) or ULK1 shRNA transfected-SW-1990 cells before and after hypoxia stimuli, respectively. Scale bar = 20 μm. (C) Coimmunoprecipitation assay detecting the abundance of YAP Ser phosphorylation in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (D) Cellular ubiquitination assays comparing the poly-Ub levels of YAP in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (E) CHX pulse-chase experiments comparing the turnover of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli in the presence or absence of 20 μg/mL CHX treatment for the indicated times, respectively. (F) Cellular ubiquitination assays evaluating the poly-Ub levels of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli. (G) Western blotting examining the expression levels of YAP in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (H) ChIP analysis for YAP binding to PKM2 gene promoter in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution using the indicated antibodies. (I) Western-blotting analyses assessing the levels of PKM2 protein in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (J) RT-qPCR analyses of PKM2 gene expression in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Luciferase-reporter PKM2 promoter activity analysis of YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.
    Figure Legend Snippet: Ser227 phosphorylation stabilizes YAP and augments PKM2 transcription upon hypoxia. (A) Representative immunfluorescence images of ULK1 nuclear localization in SW-1990 cells before and after hypoxia stimuli. Scale bar = 20 μm. (B) Representative immunfluorescence images detecting the localization of ULK1 and YAP in YAP knockout (KO) or ULK1 shRNA transfected-SW-1990 cells before and after hypoxia stimuli, respectively. Scale bar = 20 μm. (C) Coimmunoprecipitation assay detecting the abundance of YAP Ser phosphorylation in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (D) Cellular ubiquitination assays comparing the poly-Ub levels of YAP in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (E) CHX pulse-chase experiments comparing the turnover of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli in the presence or absence of 20 μg/mL CHX treatment for the indicated times, respectively. (F) Cellular ubiquitination assays evaluating the poly-Ub levels of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli. (G) Western blotting examining the expression levels of YAP in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (H) ChIP analysis for YAP binding to PKM2 gene promoter in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution using the indicated antibodies. (I) Western-blotting analyses assessing the levels of PKM2 protein in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (J) RT-qPCR analyses of PKM2 gene expression in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Luciferase-reporter PKM2 promoter activity analysis of YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Techniques Used: Knock-Out, shRNA, Transfection, Co-Immunoprecipitation Assay, Pulse Chase, Mutagenesis, Western Blot, Expressing, Binding Assay, Quantitative RT-PCR, Luciferase, Activity Assay

    ULK1/2-YAP axis contributes to hypoxic glycolysis and tumorigenesis of PDAC cells. (A) Western-blotting analyses detecting the levels of ULK1, ULK2 and Flag protein in ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression. (B and C) Glucose consumption (B) or lactate production (C) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D-G) Colony formation assays (D and E) and Ki-67 staining analyses (F and G) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (H-J) Tumor volume (H), weight (I) and representative images (J) of xenografts excised from the tumor-bearing mice. The ULK1/2 shRNA-transfected SW-1990 cells (1×10 7 ) with or without Flag-tagged wild-type YAP expression were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus ULK1/2.shRNA. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Western-blotting analyses determining the levels of Flag protein in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (L and M) Glucose consumption (L) or lactate production (M) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (N-Q) Colony formation assays (N and O) and Ki-67 staining analyses (P and Q) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (R-T) Tumor volume (R), weight (S) and representative images (T) of xenografts excised from the tumor-bearing mice. The YAP knockout (KO) SW-1990 cells (1×10 7 ) with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus YAP KO+WT. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.
    Figure Legend Snippet: ULK1/2-YAP axis contributes to hypoxic glycolysis and tumorigenesis of PDAC cells. (A) Western-blotting analyses detecting the levels of ULK1, ULK2 and Flag protein in ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression. (B and C) Glucose consumption (B) or lactate production (C) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D-G) Colony formation assays (D and E) and Ki-67 staining analyses (F and G) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (H-J) Tumor volume (H), weight (I) and representative images (J) of xenografts excised from the tumor-bearing mice. The ULK1/2 shRNA-transfected SW-1990 cells (1×10 7 ) with or without Flag-tagged wild-type YAP expression were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus ULK1/2.shRNA. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Western-blotting analyses determining the levels of Flag protein in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (L and M) Glucose consumption (L) or lactate production (M) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (N-Q) Colony formation assays (N and O) and Ki-67 staining analyses (P and Q) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (R-T) Tumor volume (R), weight (S) and representative images (T) of xenografts excised from the tumor-bearing mice. The YAP knockout (KO) SW-1990 cells (1×10 7 ) with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus YAP KO+WT. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Techniques Used: Western Blot, shRNA, Transfection, Expressing, Staining, Knock-Out, Mutagenesis

    Targeting ULK1/2 potentiates therapeutic efficacy of 2-DG and 3-BP against PDAC. (A and B) MTT assays meauring cell viability of SW-1990 cells treated with the indicated concentrations of 2-DG (A) and 3-BP (B) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C and D) Quantification of flow cytometry with Annexin-V/PI staining in SW-1990 cells treated with 25 mM 2-DG (C) and 100 μM 3-BP (D) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) Tumor volume of xenografts from the tumor-bearing mice. SW-1990 cells (S1, 1×10 7 ) were subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from seven days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. CDX, cell-derived xenograft. (F) Overall survival of mice with S1 xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (G) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (I and J). Scale bar = 100 μm. (H and I) MTT assays meauring cell viability of PDC treated with the indicated concentrations of 2-DG (H) and 3-BP (I) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (J and K) Quantification of flow cytometry with Annexin-V/PI staining in PDC treated with 25 mM 2-DG (J) and 100 μM 3-BP (K) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (L) Tumor volume of xenografts from the tumor-bearing mice. Small pieces of tumor specimens from PDAC patient were mixed with matrigel and subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from fifteen days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. PDX, patient-derived xenograft. (M) Overall survival of mice with PDX xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (N) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (L and M). Scale bar = 100 μm.
    Figure Legend Snippet: Targeting ULK1/2 potentiates therapeutic efficacy of 2-DG and 3-BP against PDAC. (A and B) MTT assays meauring cell viability of SW-1990 cells treated with the indicated concentrations of 2-DG (A) and 3-BP (B) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C and D) Quantification of flow cytometry with Annexin-V/PI staining in SW-1990 cells treated with 25 mM 2-DG (C) and 100 μM 3-BP (D) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) Tumor volume of xenografts from the tumor-bearing mice. SW-1990 cells (S1, 1×10 7 ) were subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from seven days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. CDX, cell-derived xenograft. (F) Overall survival of mice with S1 xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (G) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (I and J). Scale bar = 100 μm. (H and I) MTT assays meauring cell viability of PDC treated with the indicated concentrations of 2-DG (H) and 3-BP (I) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (J and K) Quantification of flow cytometry with Annexin-V/PI staining in PDC treated with 25 mM 2-DG (J) and 100 μM 3-BP (K) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (L) Tumor volume of xenografts from the tumor-bearing mice. Small pieces of tumor specimens from PDAC patient were mixed with matrigel and subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from fifteen days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. PDX, patient-derived xenograft. (M) Overall survival of mice with PDX xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (N) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (L and M). Scale bar = 100 μm.

    Techniques Used: Flow Cytometry, Staining, Injection, Derivative Assay

    code 8054  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc code 8054
    Code 8054, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ulk1
    TAMs-exo-derived lncRNA H19 stabilized <t>ULK1</t> in bladder cancer cells. (a) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. (b) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-overexpressed TAMs-exosome-treated BC cells after starvation for 2 h. (c) After treatment with the protein synthesis inhibitor CHX for indicated times, the protein levels of ULK1 in H19-silenced TAMs-exosome-treated T24 cells were examined by western blot. (d) Quantification of ULK1 expression in (c). (e) Western blot analysis of K48-ub levels of ULK1 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. Data shown are representative images or expressed as the mean ± SD. ∗∗ p < 0.01 compared to the control.
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Long Noncoding RNA H19 Derived from M2 Tumor-Associated Macrophages Promotes Bladder Cell Autophagy via Stabilizing ULK1"

    Article Title: Long Noncoding RNA H19 Derived from M2 Tumor-Associated Macrophages Promotes Bladder Cell Autophagy via Stabilizing ULK1

    Journal: Journal of Oncology

    doi: 10.1155/2022/3465459

    TAMs-exo-derived lncRNA H19 stabilized ULK1 in bladder cancer cells. (a) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. (b) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-overexpressed TAMs-exosome-treated BC cells after starvation for 2 h. (c) After treatment with the protein synthesis inhibitor CHX for indicated times, the protein levels of ULK1 in H19-silenced TAMs-exosome-treated T24 cells were examined by western blot. (d) Quantification of ULK1 expression in (c). (e) Western blot analysis of K48-ub levels of ULK1 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. Data shown are representative images or expressed as the mean ± SD. ∗∗ p < 0.01 compared to the control.
    Figure Legend Snippet: TAMs-exo-derived lncRNA H19 stabilized ULK1 in bladder cancer cells. (a) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. (b) Western blot analysis of protein levels of ULK1, ATG13, and ATG5 in control or H19-overexpressed TAMs-exosome-treated BC cells after starvation for 2 h. (c) After treatment with the protein synthesis inhibitor CHX for indicated times, the protein levels of ULK1 in H19-silenced TAMs-exosome-treated T24 cells were examined by western blot. (d) Quantification of ULK1 expression in (c). (e) Western blot analysis of K48-ub levels of ULK1 in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h. Data shown are representative images or expressed as the mean ± SD. ∗∗ p < 0.01 compared to the control.

    Techniques Used: Derivative Assay, Western Blot, Expressing

    TAMs-exo-derived lncRNA H19 prevents interaction between ULK1 and NEDD4L in bladder cancer cells. (a) Western blot analysis of interaction between ULK1 and NEDD4L in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h in the presence of the proteasome inhibitor mg132. (b) Western blot analysis of interaction between ULK1 and NEDD4L in control or H19-overexpressed TAMs-exosome-treated BC cells after starvation for 2 h. Data shown are representative images or expressed as the mean ± SD.
    Figure Legend Snippet: TAMs-exo-derived lncRNA H19 prevents interaction between ULK1 and NEDD4L in bladder cancer cells. (a) Western blot analysis of interaction between ULK1 and NEDD4L in control or H19-silenced TAMs-exosome-treated BC cells after starvation for 2 h in the presence of the proteasome inhibitor mg132. (b) Western blot analysis of interaction between ULK1 and NEDD4L in control or H19-overexpressed TAMs-exosome-treated BC cells after starvation for 2 h. Data shown are representative images or expressed as the mean ± SD.

    Techniques Used: Derivative Assay, Western Blot

    TAMs-exo-derived lncRNA H19 aggravates autophagy in vivo . (a) Images of representative tumors excised from mice in groups. (b, c) The volume (b) and weight (c) of xenograft tumors were measured ( N = 5 per group). (d) Western blot results of LC3, p62, and ULK1 in tumors of groups. Data shown are representative images or expressed as the mean ± SD. ∗∗∗ p < 0.001.
    Figure Legend Snippet: TAMs-exo-derived lncRNA H19 aggravates autophagy in vivo . (a) Images of representative tumors excised from mice in groups. (b, c) The volume (b) and weight (c) of xenograft tumors were measured ( N = 5 per group). (d) Western blot results of LC3, p62, and ULK1 in tumors of groups. Data shown are representative images or expressed as the mean ± SD. ∗∗∗ p < 0.001.

    Techniques Used: Derivative Assay, In Vivo, Western Blot

    ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ulk1
    <t>ULK1</t> is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3"

    Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2021/2896674

    ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Immunofluorescence, Staining, Immunohistochemistry

    ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vitro, Staining, Expressing

    JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Western Blot, Staining, Expressing

    ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining

    ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, In Vivo, Staining, Immunohistochemistry

    OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vivo, Staining, Immunohistochemistry

    Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.
    Figure Legend Snippet: Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.

    Techniques Used: Activity Assay

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    Cell Signaling Technology Inc ulk1
    <t>ULK1</t> is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
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    1) Product Images from "ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3"

    Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2021/2896674

    ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Immunofluorescence, Staining, Immunohistochemistry

    ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vitro, Staining, Expressing

    JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Western Blot, Staining, Expressing

    ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining

    ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, In Vivo, Staining, Immunohistochemistry

    OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vivo, Staining, Immunohistochemistry

    Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.
    Figure Legend Snippet: Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.

    Techniques Used: Activity Assay

    ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ulk1
    <t>ULK1</t> is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3"

    Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2021/2896674

    ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Immunofluorescence, Staining, Immunohistochemistry

    ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μ m). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μ m). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μ m). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vitro, Staining, Expressing

    JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: JNK signalling mediates the effect of ULK1. (a, b) Western blotting (a) and quantitative analysis (b) of c-Jun N-terminal kinase (JNK) signalling in RAW264.7 cells overexpressing ULK1 treated with 50 ng/ml of RANKL for 0–60 minutes. (c, d) Western blotting (c) and quantitative analysis (d) of JNK signalling in RAW264.7 cells with ULK1 knockdown treated with 50 ng/ml RANKL for 0–60 minutes. (e, f) TRAP staining (e) and quantification (f) in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 OC (scale bar, 100 μ m). (g) OC-specific gene expression in Si-NC, JNK-IN-8, and Si-ULK1 with JNK-IN-8 BMM. (h, i) Western blot (h) and quantitative analysis (i) of p-Syk and total spleen tyrosine kinase (Syk) levels in Si-NC and Si-ULK1 RAW264.7 cells. (j, k) Western blot (j) and quantitative analysis (k) of p-JNK and total JNK levels in Si-NC and Si-ULK1 cells treated with or without PRT062607. (l, m) TRAP staining (l) and quantification (m) in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607 (scale bar, 100 μ m). (n) The expression of OC-specific genes in BMM of Si-NC, Si-ULK1, and Si-ULK1 treated with PRT062607. All data are means ± SEM; ns P > 0.05. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Western Blot, Staining, Expressing

    ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μ m). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μ m). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μ m). All data are means ± SEM; ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining

    ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: ULK1 activation alleviates bone loss in vivo. (a) Schematic diagram of treatment of OVX mice. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from sham, therapy, and control mice. Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD ( n = 5). (d) H&E staining of femur sections from sham, OVX, and OVX mice treated with LYN-1604 therapy (scale bar, 100 μ m). (e) TRAP staining of femur sections from sham, OVX, and OVX with LYN-1604 therapy. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from OVX mice treated with vehicle and LYN-1604 (scale bar, 100 μ m). All data are means ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: Activation Assay, In Vivo, Staining, Immunohistochemistry

    OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
    Figure Legend Snippet: OC overexpressing ULK1 reduces bone resorption in vivo. (a) Schematic diagram of BMM transfer. (b) Representative μ CT images (scale bars, 0.5 mm). (c) Quantification of cortical bone parameters by μ CT from mice implanted with control and ULK1 overexpressing cells ( n = 8). Tb. Sp; Tb. N; Tb. Th; BV/TV; BMD. (d) H&E staining of femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (e) TRAP staining of femur sections from mice implanted with control and ULK1 overexpressing cells. The yellow arrowhead points to OC (scale bar, 100 μ m). (f) Immunohistochemistry of ULK1 in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). (g) Immunohistochemistry of p-JNK in femur sections from mice implanted with control and ULK1 overexpressing cells (scale bar, 100 μ m). All data are mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

    Techniques Used: In Vivo, Staining, Immunohistochemistry

    Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.
    Figure Legend Snippet: Schematic diagram of the inhibitory effect of ULK1 on osteoclast differentiation and bone resorption activity.

    Techniques Used: Activity Assay

    ulk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ulk1
    Knockdown of FOXK2 promoted the expression of autophagy-related protein <t>ULK1,</t> VPS34 and FOXO3. (A) Western blot detection of ULK1, VPS34 and FOXO3 in BHT-101 cells transfected with indicated plasmids. (B-D) The protein expression of ULK1 (B), VPS34 (C) and FOXO3 (D) in BHT-101 cells was visualized via quantitative analysis. * P < 0.05. (E) Western blot detection of ULK1, VPS34 and FOXO3 in BCPAP cells transfected with indicated plasmids. (F-H) The protein expression of ULK1 (F), VPS34 (G) and FOXO3 (H) in BCPAP cells was visualized via quantitative analysis. ** P < 0.01. (I) Western blot detection of FOXK2, ULK1, VPS34, FOXO3 and p62 in BCPAP cells transfected with indicated plasmids and siRNA.
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ulk1/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ulk1 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "FOXK2 promotes the proliferation of papillary thyroid cancer cell by down-regulating autophagy"

    Article Title: FOXK2 promotes the proliferation of papillary thyroid cancer cell by down-regulating autophagy

    Journal: Journal of Cancer

    doi: 10.7150/jca.60730

    Knockdown of FOXK2 promoted the expression of autophagy-related protein ULK1, VPS34 and FOXO3. (A) Western blot detection of ULK1, VPS34 and FOXO3 in BHT-101 cells transfected with indicated plasmids. (B-D) The protein expression of ULK1 (B), VPS34 (C) and FOXO3 (D) in BHT-101 cells was visualized via quantitative analysis. * P < 0.05. (E) Western blot detection of ULK1, VPS34 and FOXO3 in BCPAP cells transfected with indicated plasmids. (F-H) The protein expression of ULK1 (F), VPS34 (G) and FOXO3 (H) in BCPAP cells was visualized via quantitative analysis. ** P < 0.01. (I) Western blot detection of FOXK2, ULK1, VPS34, FOXO3 and p62 in BCPAP cells transfected with indicated plasmids and siRNA.
    Figure Legend Snippet: Knockdown of FOXK2 promoted the expression of autophagy-related protein ULK1, VPS34 and FOXO3. (A) Western blot detection of ULK1, VPS34 and FOXO3 in BHT-101 cells transfected with indicated plasmids. (B-D) The protein expression of ULK1 (B), VPS34 (C) and FOXO3 (D) in BHT-101 cells was visualized via quantitative analysis. * P < 0.05. (E) Western blot detection of ULK1, VPS34 and FOXO3 in BCPAP cells transfected with indicated plasmids. (F-H) The protein expression of ULK1 (F), VPS34 (G) and FOXO3 (H) in BCPAP cells was visualized via quantitative analysis. ** P < 0.01. (I) Western blot detection of FOXK2, ULK1, VPS34, FOXO3 and p62 in BCPAP cells transfected with indicated plasmids and siRNA.

    Techniques Used: Expressing, Western Blot, Transfection

    ulk1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc ulk1
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ulk1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc ulk1
    Changes of AMPK, mTOR, and <t>ULK1</t> expression during late exercise preconditioning. (a–e) Images and quantitative analysis of AMPK, AMPK α Thr172 , mTOR, ULK1, and ULK1 Ser757 in rat myocardium by western blot assay. (f) Quantitative analysis of ULK1 Ser757 /ULK1. ∗ P < 0.05 vs. C. (g) Autophagosomes and lysosomes were observed from these groups by TEM. (×5,000), bar = 1.0 μ m.
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ulk1/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
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    ulk1 - by Bioz Stars, 2023-02
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    1) Product Images from "Late Exercise Preconditioning Promotes Autophagy against Exhaustive Exercise-Induced Myocardial Injury through the Activation of the AMPK-mTOR-ULK1 Pathway"

    Article Title: Late Exercise Preconditioning Promotes Autophagy against Exhaustive Exercise-Induced Myocardial Injury through the Activation of the AMPK-mTOR-ULK1 Pathway

    Journal: BioMed Research International

    doi: 10.1155/2019/5697380

    Changes of AMPK, mTOR, and ULK1 expression during late exercise preconditioning. (a–e) Images and quantitative analysis of AMPK, AMPK α Thr172 , mTOR, ULK1, and ULK1 Ser757 in rat myocardium by western blot assay. (f) Quantitative analysis of ULK1 Ser757 /ULK1. ∗ P < 0.05 vs. C. (g) Autophagosomes and lysosomes were observed from these groups by TEM. (×5,000), bar = 1.0 μ m.
    Figure Legend Snippet: Changes of AMPK, mTOR, and ULK1 expression during late exercise preconditioning. (a–e) Images and quantitative analysis of AMPK, AMPK α Thr172 , mTOR, ULK1, and ULK1 Ser757 in rat myocardium by western blot assay. (f) Quantitative analysis of ULK1 Ser757 /ULK1. ∗ P < 0.05 vs. C. (g) Autophagosomes and lysosomes were observed from these groups by TEM. (×5,000), bar = 1.0 μ m.

    Techniques Used: Expressing, Western Blot

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    Cell Signaling Technology Inc ulk1
    Ulk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti ulk1
    Correlation between <t>ULK1/2,</t> YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.
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    Cell Signaling Technology Inc code 8054
    Correlation between <t>ULK1/2,</t> YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.
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    Image Search Results


    Correlation between ULK1/2, YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: Correlation between ULK1/2, YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Immunohistochemistry, Expressing, Staining, Western Blot

    Kinase activity of ULK1/2 is instrumental for hypoxic glycolysis mediated by PKM2 independent of BNIP3. (A) Western-blotting comparing the levels of ULK1 and ULK2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia, respectively. GAPDH was used as internal control of cytoplasmic lysates. Scr, scrambled shRNA; WB, western-blotting. (B and C) Glucose consumption (B) or lactate production (C) of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D and E) Glucose consumption (D) or lactate production (E) of SW-1990 cells treated with SBI-0206965 (SBI) at the indicated concentrations under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus D. D, DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Western-blotting detecting the amount of ULK1, ULK2 and BNIP3 expression in SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (G and H) Glucose consumption (G) or lactate production (H) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) Western-blotting assessing the amount of BNIP3 expression in SW-1990 cells with BNIP3 shRNA (sh.BNIP3) transfection. (J and K) Glucose consumption (J) or lactate production (K) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided Student's t test was used to calculate the P value. (L) Western-blotting detecting the amount of ULK1, ULK2 and PKM2 expression in SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (M and N) Glucose consumption (M) or lactate production (N) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. N.S., no significant. (O) Western-blotting examining the amount of PKM2 expression in SW-1990 cells with PKM2 shRNA (sh.PKM2) transfection. (P and Q) Glucose consumption (P) or lactate production (Q) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided Student's t test was used to calculate the P value.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: Kinase activity of ULK1/2 is instrumental for hypoxic glycolysis mediated by PKM2 independent of BNIP3. (A) Western-blotting comparing the levels of ULK1 and ULK2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia, respectively. GAPDH was used as internal control of cytoplasmic lysates. Scr, scrambled shRNA; WB, western-blotting. (B and C) Glucose consumption (B) or lactate production (C) of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D and E) Glucose consumption (D) or lactate production (E) of SW-1990 cells treated with SBI-0206965 (SBI) at the indicated concentrations under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus D. D, DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Western-blotting detecting the amount of ULK1, ULK2 and BNIP3 expression in SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (G and H) Glucose consumption (G) or lactate production (H) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) Western-blotting assessing the amount of BNIP3 expression in SW-1990 cells with BNIP3 shRNA (sh.BNIP3) transfection. (J and K) Glucose consumption (J) or lactate production (K) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided Student's t test was used to calculate the P value. (L) Western-blotting detecting the amount of ULK1, ULK2 and PKM2 expression in SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (M and N) Glucose consumption (M) or lactate production (N) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. N.S., no significant. (O) Western-blotting examining the amount of PKM2 expression in SW-1990 cells with PKM2 shRNA (sh.PKM2) transfection. (P and Q) Glucose consumption (P) or lactate production (Q) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided Student's t test was used to calculate the P value.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Activity Assay, Western Blot, Expressing, Transfection, shRNA

    YAP transactivates PKM2 in an ULK1/2-dependent fashion under hypoxia. (A) Western-blotting determining the amount of PKM2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Scr, scrambled shRNA. (B) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C) Representative IHC staining images (c1) and quantification (c2) for PKM2 staining in the xenografts from mice injected with SW-1990 cells expressing ULK1, ULK2 or ULK1/2 shRNA ( n =6). Scale bar = 50 μm. (D) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ChIP analysis for YAP and HIF-1α binding to PKM2 gene promoter in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of hypoxia, respectively. (F) Coimmunoprecipitation assay testing the interaction between nuclear YAP and HIF-1α in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. (G) RT-qPCR analyses of YAP1 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 and *** P <0.001 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Western-blotting comparing the abundance of PKM2 expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia. (I) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value. (J) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: YAP transactivates PKM2 in an ULK1/2-dependent fashion under hypoxia. (A) Western-blotting determining the amount of PKM2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Scr, scrambled shRNA. (B) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C) Representative IHC staining images (c1) and quantification (c2) for PKM2 staining in the xenografts from mice injected with SW-1990 cells expressing ULK1, ULK2 or ULK1/2 shRNA ( n =6). Scale bar = 50 μm. (D) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ChIP analysis for YAP and HIF-1α binding to PKM2 gene promoter in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of hypoxia, respectively. (F) Coimmunoprecipitation assay testing the interaction between nuclear YAP and HIF-1α in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. (G) RT-qPCR analyses of YAP1 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 and *** P <0.001 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Western-blotting comparing the abundance of PKM2 expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia. (I) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value. (J) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Western Blot, Expressing, Transfection, shRNA, Quantitative RT-PCR, Immunohistochemistry, Staining, Injection, Luciferase, Activity Assay, Binding Assay, Co-Immunoprecipitation Assay, Knock-Out

    ULK1 translocates into nucleus and phosphorylates YAP at Ser227 during hypoxia, which leads to YAP stabilization. (A) CHX pulse-chase experiments comparing the turnover of YAP protein in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of 20 μg/mL CHX treatment for the indicated times under hypoxia, respectively. (B) Western blotting analyses testing abundance of YAP protein in the hypoxia-stimulated SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of 10 μmol/L MG132 treatment, respectively. (C) Cellular ubiquitination assays examining the poly-Ub levels of YAP in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. PD, pull-down; Ni-NTA, Ni 2+ -nitrilotriacetic acid (NTA). (D) Coimmunoprecipitation assay evaluating the interaction between ULK1 and nuclear YAP in SW-1990 cells with or without hypoxia stimuli. (E) His-pulldown assay for determination of ULK1-YAP interaction with mixing purified His-tagged ULK1 immobilized on Ni 2+ -nitrilotriacetic acid (NTA)-sepharose beads and nuclear lysates from hypoxia-stimulated SW-1990 cells expressing Flag-tagged wild-type YAP followed by WB analyses of proteins on beads with an anti-Flag antibody. PD, pull-down. (F) Sequence alignment of the evolutionarily conserved ULK1-phosphorylating motif 224 MMN*SA 228 peptide in amino acid sequence of YAP protein among human and other primates. (G) Left panel: western-blotting examining the levels of ULK1 protein in the SW-1990 cells transfected with ULK1 shRNA. Right panel: coimmunoprecipitation assay detecting the abundance of Flag-tagged wild-type YAP phosphorylation in hypoxia-stimulated SW-1990 cells in the presence or absence of ULK1 shRNA transfection with an anti-phospho-serine antibody. (H) Coimmunoprecipitation assay comparing the levels of Flag-tagged wild-type YAP (WT), mutant YAP Ser227A (S227A) and Ser227E (S227E) phosphorylation in hypoxia-stimulated SW-1990 cells with an anti-phospho-serine antibody. (I) In vitro kinase assay with mixing His-tagged ULK1 protein and IPs of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) followed by WB analyses with an anti-phospho-serine antibody. (J) Schematic defining the role of ULK1/2 in Ser227 phosphorylation and stabilization of nuclear YAP during PDAC development in response to hypoxia sensing.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: ULK1 translocates into nucleus and phosphorylates YAP at Ser227 during hypoxia, which leads to YAP stabilization. (A) CHX pulse-chase experiments comparing the turnover of YAP protein in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of 20 μg/mL CHX treatment for the indicated times under hypoxia, respectively. (B) Western blotting analyses testing abundance of YAP protein in the hypoxia-stimulated SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of 10 μmol/L MG132 treatment, respectively. (C) Cellular ubiquitination assays examining the poly-Ub levels of YAP in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. PD, pull-down; Ni-NTA, Ni 2+ -nitrilotriacetic acid (NTA). (D) Coimmunoprecipitation assay evaluating the interaction between ULK1 and nuclear YAP in SW-1990 cells with or without hypoxia stimuli. (E) His-pulldown assay for determination of ULK1-YAP interaction with mixing purified His-tagged ULK1 immobilized on Ni 2+ -nitrilotriacetic acid (NTA)-sepharose beads and nuclear lysates from hypoxia-stimulated SW-1990 cells expressing Flag-tagged wild-type YAP followed by WB analyses of proteins on beads with an anti-Flag antibody. PD, pull-down. (F) Sequence alignment of the evolutionarily conserved ULK1-phosphorylating motif 224 MMN*SA 228 peptide in amino acid sequence of YAP protein among human and other primates. (G) Left panel: western-blotting examining the levels of ULK1 protein in the SW-1990 cells transfected with ULK1 shRNA. Right panel: coimmunoprecipitation assay detecting the abundance of Flag-tagged wild-type YAP phosphorylation in hypoxia-stimulated SW-1990 cells in the presence or absence of ULK1 shRNA transfection with an anti-phospho-serine antibody. (H) Coimmunoprecipitation assay comparing the levels of Flag-tagged wild-type YAP (WT), mutant YAP Ser227A (S227A) and Ser227E (S227E) phosphorylation in hypoxia-stimulated SW-1990 cells with an anti-phospho-serine antibody. (I) In vitro kinase assay with mixing His-tagged ULK1 protein and IPs of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) followed by WB analyses with an anti-phospho-serine antibody. (J) Schematic defining the role of ULK1/2 in Ser227 phosphorylation and stabilization of nuclear YAP during PDAC development in response to hypoxia sensing.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Pulse Chase, Transfection, shRNA, Western Blot, Co-Immunoprecipitation Assay, Purification, Expressing, Sequencing, Mutagenesis, In Vitro, Kinase Assay

    Ser227 phosphorylation stabilizes YAP and augments PKM2 transcription upon hypoxia. (A) Representative immunfluorescence images of ULK1 nuclear localization in SW-1990 cells before and after hypoxia stimuli. Scale bar = 20 μm. (B) Representative immunfluorescence images detecting the localization of ULK1 and YAP in YAP knockout (KO) or ULK1 shRNA transfected-SW-1990 cells before and after hypoxia stimuli, respectively. Scale bar = 20 μm. (C) Coimmunoprecipitation assay detecting the abundance of YAP Ser phosphorylation in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (D) Cellular ubiquitination assays comparing the poly-Ub levels of YAP in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (E) CHX pulse-chase experiments comparing the turnover of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli in the presence or absence of 20 μg/mL CHX treatment for the indicated times, respectively. (F) Cellular ubiquitination assays evaluating the poly-Ub levels of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli. (G) Western blotting examining the expression levels of YAP in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (H) ChIP analysis for YAP binding to PKM2 gene promoter in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution using the indicated antibodies. (I) Western-blotting analyses assessing the levels of PKM2 protein in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (J) RT-qPCR analyses of PKM2 gene expression in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Luciferase-reporter PKM2 promoter activity analysis of YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: Ser227 phosphorylation stabilizes YAP and augments PKM2 transcription upon hypoxia. (A) Representative immunfluorescence images of ULK1 nuclear localization in SW-1990 cells before and after hypoxia stimuli. Scale bar = 20 μm. (B) Representative immunfluorescence images detecting the localization of ULK1 and YAP in YAP knockout (KO) or ULK1 shRNA transfected-SW-1990 cells before and after hypoxia stimuli, respectively. Scale bar = 20 μm. (C) Coimmunoprecipitation assay detecting the abundance of YAP Ser phosphorylation in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (D) Cellular ubiquitination assays comparing the poly-Ub levels of YAP in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (E) CHX pulse-chase experiments comparing the turnover of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli in the presence or absence of 20 μg/mL CHX treatment for the indicated times, respectively. (F) Cellular ubiquitination assays evaluating the poly-Ub levels of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli. (G) Western blotting examining the expression levels of YAP in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (H) ChIP analysis for YAP binding to PKM2 gene promoter in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution using the indicated antibodies. (I) Western-blotting analyses assessing the levels of PKM2 protein in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (J) RT-qPCR analyses of PKM2 gene expression in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Luciferase-reporter PKM2 promoter activity analysis of YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Knock-Out, shRNA, Transfection, Co-Immunoprecipitation Assay, Pulse Chase, Mutagenesis, Western Blot, Expressing, Binding Assay, Quantitative RT-PCR, Luciferase, Activity Assay

    ULK1/2-YAP axis contributes to hypoxic glycolysis and tumorigenesis of PDAC cells. (A) Western-blotting analyses detecting the levels of ULK1, ULK2 and Flag protein in ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression. (B and C) Glucose consumption (B) or lactate production (C) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D-G) Colony formation assays (D and E) and Ki-67 staining analyses (F and G) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (H-J) Tumor volume (H), weight (I) and representative images (J) of xenografts excised from the tumor-bearing mice. The ULK1/2 shRNA-transfected SW-1990 cells (1×10 7 ) with or without Flag-tagged wild-type YAP expression were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus ULK1/2.shRNA. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Western-blotting analyses determining the levels of Flag protein in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (L and M) Glucose consumption (L) or lactate production (M) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (N-Q) Colony formation assays (N and O) and Ki-67 staining analyses (P and Q) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (R-T) Tumor volume (R), weight (S) and representative images (T) of xenografts excised from the tumor-bearing mice. The YAP knockout (KO) SW-1990 cells (1×10 7 ) with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus YAP KO+WT. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: ULK1/2-YAP axis contributes to hypoxic glycolysis and tumorigenesis of PDAC cells. (A) Western-blotting analyses detecting the levels of ULK1, ULK2 and Flag protein in ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression. (B and C) Glucose consumption (B) or lactate production (C) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D-G) Colony formation assays (D and E) and Ki-67 staining analyses (F and G) of ULK1/2 shRNA-transfected SW-1990 cells with or without Flag-tagged wild-type YAP expression under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (H-J) Tumor volume (H), weight (I) and representative images (J) of xenografts excised from the tumor-bearing mice. The ULK1/2 shRNA-transfected SW-1990 cells (1×10 7 ) with or without Flag-tagged wild-type YAP expression were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus ULK1/2.shRNA. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Western-blotting analyses determining the levels of Flag protein in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (L and M) Glucose consumption (L) or lactate production (M) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (N-Q) Colony formation assays (N and O) and Ki-67 staining analyses (P and Q) of YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution under hypoxia. Data are expressed as mean ± s.d. of five independent experiments. * P <0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Scale bar = 100 μm. (R-T) Tumor volume (R), weight (S) and representative images (T) of xenografts excised from the tumor-bearing mice. The YAP knockout (KO) SW-1990 cells (1×10 7 ) with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution were subcutaneously inoculated into the right flank of nude mice (n=6). The mice were sacrificed and the tumors were excised and measured on day 27. Data are presented as mean ± s.d. * P <0.05 versus YAP KO+WT. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Western Blot, shRNA, Transfection, Expressing, Staining, Knock-Out, Mutagenesis

    Targeting ULK1/2 potentiates therapeutic efficacy of 2-DG and 3-BP against PDAC. (A and B) MTT assays meauring cell viability of SW-1990 cells treated with the indicated concentrations of 2-DG (A) and 3-BP (B) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C and D) Quantification of flow cytometry with Annexin-V/PI staining in SW-1990 cells treated with 25 mM 2-DG (C) and 100 μM 3-BP (D) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) Tumor volume of xenografts from the tumor-bearing mice. SW-1990 cells (S1, 1×10 7 ) were subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from seven days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. CDX, cell-derived xenograft. (F) Overall survival of mice with S1 xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (G) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (I and J). Scale bar = 100 μm. (H and I) MTT assays meauring cell viability of PDC treated with the indicated concentrations of 2-DG (H) and 3-BP (I) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (J and K) Quantification of flow cytometry with Annexin-V/PI staining in PDC treated with 25 mM 2-DG (J) and 100 μM 3-BP (K) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (L) Tumor volume of xenografts from the tumor-bearing mice. Small pieces of tumor specimens from PDAC patient were mixed with matrigel and subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from fifteen days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. PDX, patient-derived xenograft. (M) Overall survival of mice with PDX xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (N) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (L and M). Scale bar = 100 μm.

    Journal: International Journal of Biological Sciences

    Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

    doi: 10.7150/ijbs.60018

    Figure Lengend Snippet: Targeting ULK1/2 potentiates therapeutic efficacy of 2-DG and 3-BP against PDAC. (A and B) MTT assays meauring cell viability of SW-1990 cells treated with the indicated concentrations of 2-DG (A) and 3-BP (B) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C and D) Quantification of flow cytometry with Annexin-V/PI staining in SW-1990 cells treated with 25 mM 2-DG (C) and 100 μM 3-BP (D) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) Tumor volume of xenografts from the tumor-bearing mice. SW-1990 cells (S1, 1×10 7 ) were subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from seven days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. CDX, cell-derived xenograft. (F) Overall survival of mice with S1 xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (G) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (I and J). Scale bar = 100 μm. (H and I) MTT assays meauring cell viability of PDC treated with the indicated concentrations of 2-DG (H) and 3-BP (I) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 versus DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (J and K) Quantification of flow cytometry with Annexin-V/PI staining in PDC treated with 25 mM 2-DG (J) and 100 μM 3-BP (K) with or without SBI-0206965 (SBI) administration. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (L) Tumor volume of xenografts from the tumor-bearing mice. Small pieces of tumor specimens from PDAC patient were mixed with matrigel and subcutaneously inoculated into the right flank of nude mice. 2-DG or 3-BP in combination with or without SBI-0206965 (SBI) were intraperitoneally injected into nude mice for 5 day-intervals from fifteen days after inoculation. Data are presented as mean ± s.d. * P <0.05 versus 2-DG or 3-BP. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. PDX, patient-derived xenograft. (M) Overall survival of mice with PDX xenografts treated with 2-DG or 3-BP in combination with or without SBI-0206965 (SBI). Log-rank test was used to calculate the P value. (N) Representative immunfluorescence images for Ki-67 (red) and cleaved caspase-3 (cCASP3, green) staining for xenografts as in (L and M). Scale bar = 100 μm.

    Article Snippet: Primary antibodies for western-blotting, co-immunoprecipitation (co-IP), in vitro kinase and cellular ubiquitination assays are as the followings: anti-ULK1 (1:1000 dilution, Cell Signaling Technology, cat#8054), anti-ULK2 (1:1000 dilution, Abcam, cat#ab97695), anti-ULK3 (1:1000 dilution, Abcam, cat#ab219264), anti-HIF-1α (1:1000 dilution, Cell Signaling Technology, cat#36169), anti-BNIP3 (1:1000 dilution, Cell Signaling Technology, cat#44060), anti-PKM2 (1:1000 dilution, Abcam, cat#ab137852), anti-PKM1 (1:1000 dilution, Cell Signaling Technology, cat#7067), anti-YAP (1:1000 dilution, Cell Signaling Technology, cat#14074), anti-phospho-serine (1:500 dilution, Santa Cruz, cat# sc-81514), anti-K48-Ub (1:1000 dilution, Cell Signaling Technology, cat#12805S), anti-K63-Ub (1:1000 dilution, Cell Signaling Technology, cat#12930S), anti-Flag (1:1000 dilution, Biosynthesis, cat#bs-0965R), anti-Lamin B (1:1500 dilution, Cell Signaling Technology, cat#13435), anti-GAPDH (1:1500 dilution, Beyotime, cat#AF1186) and anti-β-actin (1:1500 dilution, Beyotime, cat#AF0003).

    Techniques: Flow Cytometry, Staining, Injection, Derivative Assay