44849 Search Results


nr 044849 clostridium thermobutyricum strain jw171k  (ATCC)
86
ATCC nr 044849 clostridium thermobutyricum strain jw171k
Nr 044849 Clostridium Thermobutyricum Strain Jw171k, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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type strain c atypicum dsm 44849  (DSMZ)
86
DSMZ type strain c atypicum dsm 44849
Type Strain C Atypicum Dsm 44849, supplied by DSMZ, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ulk1 sirna  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology ulk1 sirna
IL4 promotes the formation and activation of the autophagy-specific PtdIns3K complex in murine primary B cells. (A) Murine splenic B cells were treated with IL4 for different times (2, 4 and 6 h). The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (B) Murine splenic B cells were treated with IL4 in the presence or absence of the JAK3 inhibitor JANEX-1 for 2 h. The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (C to E) Splenic B cells were treated with IL4 in the presence or absence of JANEX-1, intracellular accumulation of PtdIns3P was measured by flow cytometry (C). Bar graphs (mean ± SEM) represent the percentage of PtdIns3P+ B cells (D) and intracellular PtdIns3P MFI (mean fluorescence intensity) (E). Iso, isotype control antibody. *, P<0.05; **, P<0.01; ns, not statistically significant; determined by the Student t test. Data are representative of 3 independent experiments. (F) The location of cytoplasmic PtdIns3P and LC3B puncta in B cells after IL4 treatment was observed by confocal microscopy. DAPI was used to denote the cell nucleus. Data are representative of 2 independent experiments. (G) Becn1 silencing or <t>Ulk1</t> silencing was performed in murine primary B cells, respectively, and NC (negative control) <t>siRNA</t> was used as control siRNA. Then B cells were treated with IL4 for 24 h, CQ was added for the last 3 h. The accumulation of LC3B-II was assessed by immunoblotting of whole cell lysates. Bar graphs (mean ± SEM) represent ACTB-normalized LC3B-II density values; *, P<0.05; ns, not statistically significant;determined by the Student t test. Data are representative of 2 independent experiments.
Ulk1 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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short hairpin rna shrna  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology short hairpin rna shrna
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 <t>shRNA</t> 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.
Short Hairpin Rna Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


IL4 promotes the formation and activation of the autophagy-specific PtdIns3K complex in murine primary B cells. (A) Murine splenic B cells were treated with IL4 for different times (2, 4 and 6 h). The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (B) Murine splenic B cells were treated with IL4 in the presence or absence of the JAK3 inhibitor JANEX-1 for 2 h. The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (C to E) Splenic B cells were treated with IL4 in the presence or absence of JANEX-1, intracellular accumulation of PtdIns3P was measured by flow cytometry (C). Bar graphs (mean ± SEM) represent the percentage of PtdIns3P+ B cells (D) and intracellular PtdIns3P MFI (mean fluorescence intensity) (E). Iso, isotype control antibody. *, P<0.05; **, P<0.01; ns, not statistically significant; determined by the Student t test. Data are representative of 3 independent experiments. (F) The location of cytoplasmic PtdIns3P and LC3B puncta in B cells after IL4 treatment was observed by confocal microscopy. DAPI was used to denote the cell nucleus. Data are representative of 2 independent experiments. (G) Becn1 silencing or Ulk1 silencing was performed in murine primary B cells, respectively, and NC (negative control) siRNA was used as control siRNA. Then B cells were treated with IL4 for 24 h, CQ was added for the last 3 h. The accumulation of LC3B-II was assessed by immunoblotting of whole cell lysates. Bar graphs (mean ± SEM) represent ACTB-normalized LC3B-II density values; *, P<0.05; ns, not statistically significant;determined by the Student t test. Data are representative of 2 independent experiments.

Journal: Autophagy

Article Title: IL4 (interleukin 4) induces autophagy in B cells leading to exacerbated asthma

doi: 10.1080/15548627.2017.1421884

Figure Lengend Snippet: IL4 promotes the formation and activation of the autophagy-specific PtdIns3K complex in murine primary B cells. (A) Murine splenic B cells were treated with IL4 for different times (2, 4 and 6 h). The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (B) Murine splenic B cells were treated with IL4 in the presence or absence of the JAK3 inhibitor JANEX-1 for 2 h. The binding of BECN1 to PIK3C3, ATG14 and PIK3R4 was analyzed by CoIP with anti-BECN1 antibody. WCL, whole cell lysates. Data are representative of 3 independent experiments. (C to E) Splenic B cells were treated with IL4 in the presence or absence of JANEX-1, intracellular accumulation of PtdIns3P was measured by flow cytometry (C). Bar graphs (mean ± SEM) represent the percentage of PtdIns3P+ B cells (D) and intracellular PtdIns3P MFI (mean fluorescence intensity) (E). Iso, isotype control antibody. *, P<0.05; **, P<0.01; ns, not statistically significant; determined by the Student t test. Data are representative of 3 independent experiments. (F) The location of cytoplasmic PtdIns3P and LC3B puncta in B cells after IL4 treatment was observed by confocal microscopy. DAPI was used to denote the cell nucleus. Data are representative of 2 independent experiments. (G) Becn1 silencing or Ulk1 silencing was performed in murine primary B cells, respectively, and NC (negative control) siRNA was used as control siRNA. Then B cells were treated with IL4 for 24 h, CQ was added for the last 3 h. The accumulation of LC3B-II was assessed by immunoblotting of whole cell lysates. Bar graphs (mean ± SEM) represent ACTB-normalized LC3B-II density values; *, P<0.05; ns, not statistically significant;determined by the Student t test. Data are representative of 2 independent experiments.

Article Snippet: Protein A/G plus-agarose (Santa Cruz Biotechnology, sc-2003), normal rabbit IgG (Santa Cruz Biotechnology, sc-2027), normal mouse IgG2a (Santa Cruz Biotechnology, sc-3878), and BECN1 (E8, Santa Cruz Biotechnology, sc-48341) were used in CoIP assays. siRNA silencing Murine Becn1 siRNA and Ulk1 siRNA were purchased from SyngenTech company.

Techniques: Activation Assay, Binding Assay, Flow Cytometry, Fluorescence, Confocal Microscopy, Negative Control, 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: Commercial short hairpin RNA (shRNA) targeting ULK1 (sc-44182-SH), ULK2 (sc-44183-SH) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA).

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

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: Commercial short hairpin RNA (shRNA) targeting ULK1 (sc-44182-SH), ULK2 (sc-44183-SH) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Transfection, shRNA, Quantitative RT-PCR, Gene Expression, 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: Commercial short hairpin RNA (shRNA) targeting ULK1 (sc-44182-SH), ULK2 (sc-44183-SH) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Pulse Chase, Transfection, shRNA, Western Blot, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Purification, Expressing, Sequencing, Phospho-proteomics, 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: Commercial short hairpin RNA (shRNA) targeting ULK1 (sc-44182-SH), ULK2 (sc-44183-SH) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Knock-Out, shRNA, Transfection, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Pulse Chase, Mutagenesis, Western Blot, Expressing, Binding Assay, Quantitative RT-PCR, Gene Expression, 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: Commercial short hairpin RNA (shRNA) targeting ULK1 (sc-44182-SH), ULK2 (sc-44183-SH) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA).

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