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  • 99
    Thermo Fisher β mercaptoethanol
    β Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti beta actin antibody
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β actin
    FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and <t>β-actin</t> was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P
    β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 51048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin
    Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with <t>β-actin</t> protein. Data are expressed as means ± SD ( n = 6 sample per group). P
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 38293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β actin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. <t>β-Actin</t> was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam β actin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. <t>β-Actin</t> was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 25356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 67284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β actin antibody
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 9783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti β actin
    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. <t>β-actin</t> was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P
    Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β catenin
    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). <t>β-catenin</t> immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.
    β Catenin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti β actin
    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). <t>β-catenin</t> immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.
    Mouse Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti β actin
    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). <t>β-catenin</t> immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.
    Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson β catenin
    Model of single <t>β-catenin</t> mutation and interaction with E-cadherin in the small intestine and colon A, B Model of a single activating β-catenin mutation in the murine small intestine and the colon. In contrast to the small intestine (A), the increased levels of E-cadherin in the colon complexed with mutant β-catenin prevent its accumulation in the nucleus (B).
    β Catenin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 6806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β actin
    Model of single <t>β-catenin</t> mutation and interaction with E-cadherin in the small intestine and colon A, B Model of a single activating β-catenin mutation in the murine small intestine and the colon. In contrast to the small intestine (A), the increased levels of E-cadherin in the colon complexed with mutant β-catenin prevent its accumulation in the nucleus (B).
    β Actin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β catenin
    Inactivation of <t>β-catenin</t> results in aberrant expression of SOX9 in a subset of airway epithelial cells Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) and control β-catenin fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for SOX9, SOX2 (A) and β-catenin (B). β-catenin-deficient embryos show aberrant expression of SOX9 in airway epithelium, which was identified by SOX2 (A–B). Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. Abbreviations: Br, bronchi; Cart, cartilage. Magnifications: main images, ×400; inserts, ×800.
    β Catenin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 5488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti beta tubulin antibody
    Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S and Tom20 C21S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S and Tom20 C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO 4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO 4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO 4 -induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO 4 -induced cytochrome c release detected in the cytosol fraction was diminished ( d ), the cleavage of caspase-3 and -9 was attenuated ( e ), the cell viability was rescued ( f ), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished ( g ). h Effects of the mutants Tom20 C13S and Tom20 C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO 4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. <t>Tubulin</t> was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P
    Monoclonal Anti Beta Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4819 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β tubulin
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    β Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin antibodies
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    β Actin Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti actin antibody
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    Anti Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and β-actin was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV internalization and replication in BHK-21 cells are Cav-1 independent but require plasma membrane cholesterol. (A,B) Cav-1 downregulation did not affect the internalization of FMDV. Cells were transfected with control siRNA (left panels) or Cav-1 siRNA to downregulate Cav-1 expression (right panels). The effect of siRNA on AF594–CTxB uptake was apparent (red; upper panels). FMDV (MOI 25) was allowed to bind to siRNA-transfected cells for 1 h at 4 °C and then transferred to 37 °C. After incubation for 1 h at 37 °C, the fixed cells were processed for confocal microscopy (lower panels). (B) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. The internalized FMDV were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cav-1 downregulation did not affect the synthesis of viral proteins. The siRNA-transfected cells were infected (MOI 1) for 4 h and analyzed with an anti-FMDV antibody in Western blot, and β-actin was measured as the internal control. The relative quantification of the viral proteins was determined by densitometry as shown in the histogram. (D) MβCD inhibited the internalization of FMDV, whereas Nys did not. Cells were pretreated with MβCD (10 mM) or Nys (20 μg/mL) and then infected (MOI 25) as described in the Materials and Methods. Samples were then processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in Mock-, Nys- or MβCD-treated cells. (F) Nys did not affect FMDV entry and replication. Cells were treated with Nys 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1) equivalent amounts of protein were analyzed in immunoblots, and fold induction was determined by densitometry. (G) MβCD inhibited FMDV entry and multiplication. MβCD was present only during treatment for 30 min before the infection (Pre) or 60 min after virus addition (Post). Samples were then processed for Western blot. SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Transfection, Expressing, Incubation, Confocal Microscopy, Infection, Western Blot, Standard Deviation

    FMDV entry into BHK-21 cells activates Rac1 and depends on Dynamin II. (A) Activation of Rac1 during FMDV entry. Cells were infected (MOI 10), and Rac1 activation was measured by GST-PAK1-PBD pull-down assay. Fold induction was determined by densitometry. (B,C) Rac1 Inh inhibited FMDV entry. Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rac1 Inh-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Rac1 Inh. (D–F) Pretreated cells (Rac1 Inh) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Expression of inactive form of Rac1 inhibited FMDV infection. Transfected cells with Rac1 (WT) and Rac1 (T17N) were infected (MOI 1) for 4 h at 37 °C and analyzed by Western blot. (I) Effect of Rac1 Inh on virus entry and post-entry steps. Cells were treated with Rac1 Inh 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (J) Dynasore (Dyna) inhibited FMDV entry and multiplication. Cells were treated with indicated concentrations of Dyna as in (I) and then processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV entry into BHK-21 cells activates Rac1 and depends on Dynamin II. (A) Activation of Rac1 during FMDV entry. Cells were infected (MOI 10), and Rac1 activation was measured by GST-PAK1-PBD pull-down assay. Fold induction was determined by densitometry. (B,C) Rac1 Inh inhibited FMDV entry. Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rac1 Inh-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Rac1 Inh. (D–F) Pretreated cells (Rac1 Inh) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (200 μM Rac1 Inh) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Expression of inactive form of Rac1 inhibited FMDV infection. Transfected cells with Rac1 (WT) and Rac1 (T17N) were infected (MOI 1) for 4 h at 37 °C and analyzed by Western blot. (I) Effect of Rac1 Inh on virus entry and post-entry steps. Cells were treated with Rac1 Inh 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (J) Dynasore (Dyna) inhibited FMDV entry and multiplication. Cells were treated with indicated concentrations of Dyna as in (I) and then processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Infection, Pull Down Assay, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Expressing, Transfection, Standard Deviation

    Myosin II is required for FMDV entry in BHK-21 cells. (A,B) Bleb inhibited FMDV entry. Pretreated cells (4 μM Bleb) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Bleb-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Bleb. (C–E) Pretreated cells (Bleb) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (C), Western blot (D), and TCID50 assay (E). (F) Pretreated cells (4 μM Bleb) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Bleb on virus entry and post-entry steps. Cells were treated with Bleb 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: Myosin II is required for FMDV entry in BHK-21 cells. (A,B) Bleb inhibited FMDV entry. Pretreated cells (4 μM Bleb) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Bleb-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Bleb. (C–E) Pretreated cells (Bleb) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (C), Western blot (D), and TCID50 assay (E). (F) Pretreated cells (4 μM Bleb) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Bleb on virus entry and post-entry steps. Cells were treated with Bleb 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    Pak1 is required for FMDV entry into BHK-21cells. (A,B) FMDV activated Pak1 during early post-infection. (A) Cells were infected (MOI 10), and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot analysis. The level of total Pak1 was measured as the control. Fold induction was determined by densitometry. (B) Cells were infected (MOI 25) and processed for confocal microscopy with anti-phospho-Pak1 (green), anti-FMDV (red), and DAPI (blue). (C,D) IPA-3 inhibited FMDV entry. Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (D) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or IPA-3-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( E–H ) FMDV infection was inhibited by IPA-3. (E–G) Pretreated cells (IPA-3) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (E), Western blot (F), and TCID50 assay (G). (H) Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( C ). (I) Effect of IPA-3 on virus entry and post-entry steps. Cells were treated with IPA-3 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: Pak1 is required for FMDV entry into BHK-21cells. (A,B) FMDV activated Pak1 during early post-infection. (A) Cells were infected (MOI 10), and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot analysis. The level of total Pak1 was measured as the control. Fold induction was determined by densitometry. (B) Cells were infected (MOI 25) and processed for confocal microscopy with anti-phospho-Pak1 (green), anti-FMDV (red), and DAPI (blue). (C,D) IPA-3 inhibited FMDV entry. Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (D) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or IPA-3-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( E–H ) FMDV infection was inhibited by IPA-3. (E–G) Pretreated cells (IPA-3) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR (E), Western blot (F), and TCID50 assay (G). (H) Pretreated cells (15 μM IPA-3) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( C ). (I) Effect of IPA-3 on virus entry and post-entry steps. Cells were treated with IPA-3 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Confocal Microscopy, Indirect Immunoperoxidase Assay, Reverse Transcription Polymerase Chain Reaction, TCID50 Assay, Standard Deviation

    CME is not the only pathway for FMDV internalization into BHK-21. (A–C) CPZ moderately inhibited FMDV entry and infection. (A) Cells were pretreated with CPZ (20 μM) and maintained during infection. The effect of CPZ on Alexa Fluor 594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), cells were fixed and incubated with anti-clathrin, anti-FMDV, and DAPI to stain clathrin (green), viral particles (red), and cell nuclei (blue), respectively (upper panels). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual Mock- or CPZ-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cells were treated with CPZ at 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1), equivalent amounts of protein were analyzed by Western blot with an anti-FMDV antibody, and β-actin was used as the internal control. Fold induction was determined by densitometry. (D–F) CHC downregulation moderately inhibited FMDV entry and infection. (D) Cells were transfected with control siRNA (left panels) or CHC siRNA to downregulate CHC expression (right panels). The efficiency of CHC downregulation was analyzed by immunofluorescent staining at 36 h post-transfection (green); the effect of siRNA on AF594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), siRNA-transfected cells were processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. (F) The efficiency of CHC downregulation was analyzed by immunoblotting. After 4 hpi (FMDV, MOI 1), the siRNA-transfected cells were processed for Western blot. SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: CME is not the only pathway for FMDV internalization into BHK-21. (A–C) CPZ moderately inhibited FMDV entry and infection. (A) Cells were pretreated with CPZ (20 μM) and maintained during infection. The effect of CPZ on Alexa Fluor 594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), cells were fixed and incubated with anti-clathrin, anti-FMDV, and DAPI to stain clathrin (green), viral particles (red), and cell nuclei (blue), respectively (upper panels). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual Mock- or CPZ-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C) Cells were treated with CPZ at 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. After 4 hpi (FMDV, MOI 1), equivalent amounts of protein were analyzed by Western blot with an anti-FMDV antibody, and β-actin was used as the internal control. Fold induction was determined by densitometry. (D–F) CHC downregulation moderately inhibited FMDV entry and infection. (D) Cells were transfected with control siRNA (left panels) or CHC siRNA to downregulate CHC expression (right panels). The efficiency of CHC downregulation was analyzed by immunofluorescent staining at 36 h post-transfection (green); the effect of siRNA on AF594–TF uptake was apparent (red; upper panels). After 1 hpi (FMDV, MOI 25), siRNA-transfected cells were processed for confocal microscopy as in ( A ). (E) Quantitative analysis of the internalization of FMDV in siRNA-transfected cells. (F) The efficiency of CHC downregulation was analyzed by immunoblotting. After 4 hpi (FMDV, MOI 1), the siRNA-transfected cells were processed for Western blot. SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Incubation, Staining, Western Blot, Transfection, Expressing, Confocal Microscopy, Standard Deviation

    EIPA inhibits FMDV entry into BHK-21 cells and FMDV stimulates fluid-phase uptake. (A) EIPA inhibited FMDV entry. Pretreated cells (40 μM EIPA) were infected (MOI 25) for 1 h at 37 °C and then processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or EIPA-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C–E) Pretreated cells (EIPA) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (0.2 μM EIPA) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of EIPA on virus entry and post-entry steps. Cells were treated with EIPA 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (H) FMDV stimulated fluid-phase uptake. Cells were pretreated (40 μM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with AF594-dextran for 15 min, and analyzed by FACS. (I) FMDV colocalized with dextran. FMDV (MOI 25) was allowed to bind to cells for 1 h at 4 °C. The inoculum was replaced with medium containing AF594-dextran and incubated for 15 min in 37 °C. Cells were fixed and incubated with anti-FMDV antibody (green). 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: EIPA inhibits FMDV entry into BHK-21 cells and FMDV stimulates fluid-phase uptake. (A) EIPA inhibited FMDV entry. Pretreated cells (40 μM EIPA) were infected (MOI 25) for 1 h at 37 °C and then processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or EIPA-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (C–E) Pretreated cells (EIPA) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (0.2 μM EIPA) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of EIPA on virus entry and post-entry steps. Cells were treated with EIPA 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (H) FMDV stimulated fluid-phase uptake. Cells were pretreated (40 μM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with AF594-dextran for 15 min, and analyzed by FACS. (I) FMDV colocalized with dextran. FMDV (MOI 25) was allowed to bind to cells for 1 h at 4 °C. The inoculum was replaced with medium containing AF594-dextran and incubated for 15 min in 37 °C. Cells were fixed and incubated with anti-FMDV antibody (green). 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, FACS, Incubation, Standard Deviation

    FMDV entry into BHK-21 cells depends on actin dynamics and induces actin ruffles. (A) FMDV entry induced actin ruffles. Cells were incubated with FMDV (MOI 100) for 1 h at 37 °C prior to incubation for 5 min at 37 °C, fixed, and processed for TEM. The arrow indicates a virion. The arrowhead indicates the membrane ruffle. (B,C) Jas inhibited FMDV entry. Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Jas-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Jas. (D–F) Pretreated cells (Jas) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Effect of Jas on virus entry and post-entry steps. Cells were treated with Jas 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: FMDV entry into BHK-21 cells depends on actin dynamics and induces actin ruffles. (A) FMDV entry induced actin ruffles. Cells were incubated with FMDV (MOI 100) for 1 h at 37 °C prior to incubation for 5 min at 37 °C, fixed, and processed for TEM. The arrow indicates a virion. The arrowhead indicates the membrane ruffle. (B,C) Jas inhibited FMDV entry. Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (C) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Jas-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. (D–G) FMDV infection was inhibited by Jas. (D–F) Pretreated cells (Jas) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( D ), Western blot ( E ), and TCID50 assay ( F ). (G) Pretreated cells (0.2 μM Jas) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( B ). (H) Effect of Jas on virus entry and post-entry steps. Cells were treated with Jas 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Incubation, Transmission Electron Microscopy, Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    RTKs are required for FMDV entry into BHK-21 cells. (A,B) Gen inhibited FMDV entry. Pretreated cells (60 μM Gen) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Gen-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Gen. (C–E) Pretreated cells (Gen) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (60 μM Gen) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Gen on virus entry and post-entry steps. Cells were treated with Gen 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: RTKs are required for FMDV entry into BHK-21 cells. (A,B) Gen inhibited FMDV entry. Pretreated cells (60 μM Gen) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Gen-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Gen. (C–E) Pretreated cells (Gen) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (60 μM Gen) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Gen on virus entry and post-entry steps. Cells were treated with Gen 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    PKC is required for FMDV entry and multiplication in BHK-21 cells. (A,B) Rott inhibited FMDV entry. Pretreated cells (20 μM Rott) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rott-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Rott. (C–E) Pretreated cells (Rott) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (20 μM Rott) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Rott on virus entry and post-entry steps. Cells were treated with Rott 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: PKC is required for FMDV entry and multiplication in BHK-21 cells. (A,B) Rott inhibited FMDV entry. Pretreated cells (20 μM Rott) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Rott-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–F) FMDV infection was inhibited by Rott. (C–E) Pretreated cells (Rott) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Pretreated cells (20 μM Rott) were infected (MOI 25) for 4 h at 37 °C and processed for confocal microscopy as in ( A ). (G) Effect of Rott on virus entry and post-entry steps. Cells were treated with Rott 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Standard Deviation

    PI3K is not required for FMDV entry and replication in BHK-21 cells. (A,B) Wort moderately stimulated FMDV entry. Pretreated cells (40 μM Wort) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Wort-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–E) Wort enhanced FMDV infection. Pretreated cells (Wort) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Effect of Wort on virus entry and post-entry steps. Cells were treated with Wort 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (G) PI3K downregulation moderately enhanced FMDV infection. (H) PI3K overexpression did not affect FMDV infection. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Journal: Scientific Reports

    Article Title: Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    doi: 10.1038/srep19294

    Figure Lengend Snippet: PI3K is not required for FMDV entry and replication in BHK-21 cells. (A,B) Wort moderately stimulated FMDV entry. Pretreated cells (40 μM Wort) were infected (MOI 25) for 1 h at 37 °C and processed for confocal microscopy with AF594-phalloidin (red), anti-FMDV (green), and DAPI (blue). (B) Quantitative analysis of the internalization of FMDV. The internalized FMDV were analyzed in 10 individual DMSO- or Wort-treated cells. Each experiment was performed in triplicate and the results were presented as the mean ± SD. ( C–E) Wort enhanced FMDV infection. Pretreated cells (Wort) were infected (MOI 1) for 4 h at 37 °C and analyzed by RT-PCR ( C ), Western blot ( D ), and TCID50 assay ( E ). (F) Effect of Wort on virus entry and post-entry steps. Cells were treated with Wort 30 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection. Cells were then infected (MOI 1) for 4 h at 37 °C and processed for Western blot analysis. (G) PI3K downregulation moderately enhanced FMDV infection. (H) PI3K overexpression did not affect FMDV infection. 3D, FMDV 3D; β-actin, load control; SD, standard deviation; *P

    Article Snippet: Specific antibodies against Pak1, phospho-Pak1 (Thr423), caveolin-1 (Cav-1), EEA1, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology.

    Techniques: Infection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, TCID50 Assay, Over Expression, Standard Deviation

    Eliminating expression of GLUT1 in G1fP cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP, CK18, and β-actin in lysates from G LUT 1 fl/ f l mammary cells transformed with P yVMT (G1fP cells) 72 hours after being infected with adenovirus expressing GFP (Ad-GFP) or Cre recombinase (Ad-Cre) at an MOI of 100. B–D. Uptake of 3 H-2-deoxyglucose (B) , glucose consumption ( C ), and lactate secretion ( D ) by G1fP cells previously infected with Ad-GFP or Ad-Cre as described in figure 2 . E. Proliferation is estimated by determining the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F. The concentration of ATP in the two groups of cells was determined and normalized to the DNA content of parallel monolayers. G. Lipid synthesis in G1fP cells was measured as described in figure 2 . H. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 normalized to RPL32 expression in G1fP cells that had been infected two weeks prior with Ad-GFP or Ad-Cre.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Eliminating expression of GLUT1 in G1fP cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP, CK18, and β-actin in lysates from G LUT 1 fl/ f l mammary cells transformed with P yVMT (G1fP cells) 72 hours after being infected with adenovirus expressing GFP (Ad-GFP) or Cre recombinase (Ad-Cre) at an MOI of 100. B–D. Uptake of 3 H-2-deoxyglucose (B) , glucose consumption ( C ), and lactate secretion ( D ) by G1fP cells previously infected with Ad-GFP or Ad-Cre as described in figure 2 . E. Proliferation is estimated by determining the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F. The concentration of ATP in the two groups of cells was determined and normalized to the DNA content of parallel monolayers. G. Lipid synthesis in G1fP cells was measured as described in figure 2 . H. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 normalized to RPL32 expression in G1fP cells that had been infected two weeks prior with Ad-GFP or Ad-Cre.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, In Vitro, Transformation Assay, Infection, Concentration Assay, Real-time Polymerase Chain Reaction

    Overexpression of GLUT1 in 85815GL cells accelerates tumor formation. A. 0.4 million 85815GL cells expressing control vector (V) or GLUT1 (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 3, 6, 9, 12 and 14 after implantation. B. The bioluminescence on days 3, 6, 9, 12 and 14 normalized to the day 3 value was averaged for all five mice and is presented +/−SEM. C. GLUT1 and β-actin expression evaluated in lysates of three tumor pairs by immunoblot analysis. D. GLUT1 expression evaluated by IHC in two tumor pairs. E. Low power photomicrographs of a pair of tumor sections derived from a vector control tumor (top) and a tumor overexpressing GLUT1 (bottom) immunostained for cleaved caspase 3 (left). High power photomicrographs of a pair of tumor sections immunostained for cleaved caspase 3 (middle), which are converted to binary pictures (right). F. Quantification of the number of cleaved caspase 3 positive pixels in five high power field “hot spots” in four tumors of each group. G–H. Representative high power photomicrographs of tumor sections immunostained for BrdU with hematoxylin counterstain ( G ) which is quantified ( H ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Overexpression of GLUT1 in 85815GL cells accelerates tumor formation. A. 0.4 million 85815GL cells expressing control vector (V) or GLUT1 (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 3, 6, 9, 12 and 14 after implantation. B. The bioluminescence on days 3, 6, 9, 12 and 14 normalized to the day 3 value was averaged for all five mice and is presented +/−SEM. C. GLUT1 and β-actin expression evaluated in lysates of three tumor pairs by immunoblot analysis. D. GLUT1 expression evaluated by IHC in two tumor pairs. E. Low power photomicrographs of a pair of tumor sections derived from a vector control tumor (top) and a tumor overexpressing GLUT1 (bottom) immunostained for cleaved caspase 3 (left). High power photomicrographs of a pair of tumor sections immunostained for cleaved caspase 3 (middle), which are converted to binary pictures (right). F. Quantification of the number of cleaved caspase 3 positive pixels in five high power field “hot spots” in four tumors of each group. G–H. Representative high power photomicrographs of tumor sections immunostained for BrdU with hematoxylin counterstain ( G ) which is quantified ( H ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Injection, Mouse Assay, Labeling, Immunohistochemistry, Derivative Assay

    Elimination of GLUT1 expression in G1fPt cells decreases tumor growth. A. 0.5 million G1fPt cells infected two weeks prior with adenovirus expressing GFP or Cre recombinase were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 12, 15 and 20 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 12, 15 and 20 +/− SEM for eight mice is presented with green diamonds representing “GFP” tumors and light blue squares representing “Cre” tumors. C. Expression of GLUT1 and β-actin in lysates of two tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for Ki67 with hematoxylin counterstain ( F ) which is quantified ( G ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Elimination of GLUT1 expression in G1fPt cells decreases tumor growth. A. 0.5 million G1fPt cells infected two weeks prior with adenovirus expressing GFP or Cre recombinase were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 12, 15 and 20 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 12, 15 and 20 +/− SEM for eight mice is presented with green diamonds representing “GFP” tumors and light blue squares representing “Cre” tumors. C. Expression of GLUT1 and β-actin in lysates of two tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for Ki67 with hematoxylin counterstain ( F ) which is quantified ( G ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, Infection, Injection, Mouse Assay, Labeling, Luciferase, Activity Assay, Immunohistochemistry

    GLUT1 is the most abundantly expressed GLUT family member in MMTV-c-ErbB2 tumors and a number of mouse mammary carcinoma cell lines. A. qPCR analysis to determine the expression of GLUT1–GLUT6, GLUT8–GLUT10, GLUT12–GLUT13 (eleven of the twelve mouse GLUT family transporters) relative to β-actin expression was performed with the cDNA equivalent of 50 ng RNA in six samples: mammary tumor from a MMTV-c-ErbB2 mouse (ErbB2 tumor), two different cell lines derived from these tumors (78617 and 85815), a cell line derived from a MMTV-PyVMT mouse mammary tumor (Met1), a cell line derived from a BALB/c mouse mammary tumor (4T1) and immortalized mouse mammary epithelial cells (EPH4). B. Quantitation of the number of copies of GLUT1, GLUT6, GLUT8 and GLUT9 RNA in the cDNA derived from 50 ng RNA from triplicate samples of ErbB2 Tumors, 78617 cells and 85815 cells. * indicates p

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: GLUT1 is the most abundantly expressed GLUT family member in MMTV-c-ErbB2 tumors and a number of mouse mammary carcinoma cell lines. A. qPCR analysis to determine the expression of GLUT1–GLUT6, GLUT8–GLUT10, GLUT12–GLUT13 (eleven of the twelve mouse GLUT family transporters) relative to β-actin expression was performed with the cDNA equivalent of 50 ng RNA in six samples: mammary tumor from a MMTV-c-ErbB2 mouse (ErbB2 tumor), two different cell lines derived from these tumors (78617 and 85815), a cell line derived from a MMTV-PyVMT mouse mammary tumor (Met1), a cell line derived from a BALB/c mouse mammary tumor (4T1) and immortalized mouse mammary epithelial cells (EPH4). B. Quantitation of the number of copies of GLUT1, GLUT6, GLUT8 and GLUT9 RNA in the cDNA derived from 50 ng RNA from triplicate samples of ErbB2 Tumors, 78617 cells and 85815 cells. * indicates p

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Quantitation Assay

    Overexpression of GLUT1 in 85815GL cells increases glucose transport without increasing proliferation. A. Expression of GLUT1, GFP-luciferase and β-actin in lysates of 85815GL cells expressing empty vector (V) or overexpressing GLUT1 (G1). B. Uptake of 3 H-2-deoxyglucose by cells expressing empty vector (Vec) or GLUT1 (GLUT1) in 15 minutes presented as CPM per µg DNA. C. Proliferation is estimated by determining the DNA content of cultures expressing empty vector or GLUT1 at days 0, 1, 2 and 3.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Overexpression of GLUT1 in 85815GL cells increases glucose transport without increasing proliferation. A. Expression of GLUT1, GFP-luciferase and β-actin in lysates of 85815GL cells expressing empty vector (V) or overexpressing GLUT1 (G1). B. Uptake of 3 H-2-deoxyglucose by cells expressing empty vector (Vec) or GLUT1 (GLUT1) in 15 minutes presented as CPM per µg DNA. C. Proliferation is estimated by determining the DNA content of cultures expressing empty vector or GLUT1 at days 0, 1, 2 and 3.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Over Expression, Expressing, Luciferase, Plasmid Preparation

    Reduced expression of GLUT1 in 78617GL cells decreases tumor growth. A. 0.5 million 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 2, 4, 6 and 8 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 2, 4, 6 and 8 normalized to the day 2 bioluminescence +/− SEM for five mice is presented with the black diamonds representing shCTRL tumors and grey squares representing shGLUT1 tumors. C. Expression of GLUT1 and β-actin in lysates of three tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for BrdU with hematoxylin counterstain ( F ) which is quantified ( G ).

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Reduced expression of GLUT1 in 78617GL cells decreases tumor growth. A. 0.5 million 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1) were injected into contralateral #4 mammary fat pads of athymic nude mice. Bioluminescence from the labeled tumor cells was detected on days 2, 4, 6 and 8 after implantation. The abdominal region heat map depicting luciferase activity of a representative mouse is pictured. B. The average bioluminescence on days 2, 4, 6 and 8 normalized to the day 2 bioluminescence +/− SEM for five mice is presented with the black diamonds representing shCTRL tumors and grey squares representing shGLUT1 tumors. C. Expression of GLUT1 and β-actin in lysates of three tumor pairs evaluated by immunoblot analysis. D. GLUT1 expression evaluated by IHC (with hematoxylin counterstain) in two tumor pairs. E. Representative low power photomicrographs of two pairs of tumor sections immunostained for cleaved caspase 3. F–G. Representative high power photomicrographs of a tumor pair immunostained for BrdU with hematoxylin counterstain ( F ) which is quantified ( G ).

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, shRNA, Injection, Mouse Assay, Labeling, Luciferase, Activity Assay, Immunohistochemistry

    Reduced expression of GLUT1 in 78617GL cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP-Luciferase transgene (GFP) and β-actin in lysates from 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1). B. Uptake of 3 H-2-deoxyglucose by 78617GL cells expressing control shRNA (shCTRL) or GLUT1 shRNA (shGLUT1) in 15 minutes presented as CPM per µg DNA. C–D. Glucose consumption ( C ) and lactate secretion ( D ). Glucose and lactate concentrations are normalized to the DNA content of the cultures. E. Proliferation is estimated by deteriming the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F–G. 78617GL cells were grown in soft agar for 3 weeks and colonies are pictured in F and the number of colonies per well is quantified in G . H. Quantification of BrdU positive cells in the outer edge of 12 colonies of each group. I. The concentration of ATP in the two groups of cells (lacking luciferase expression) was determined and normalized to the DNA content of parallel monolayers. J. Lipid synthesis was measured by determining the amount of 14 C in the non-aqueous chloroform fraction of methanol chloroform extracted cell lysates after 24 hour incubation with 14 C-glucose and is normalized to the DNA content of parallel samples. K. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 in 78617GL cells expressing control shRNA or GLUT1 shRNA normalized to RPL32 expression.

    Journal: PLoS ONE

    Article Title: Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0023205

    Figure Lengend Snippet: Reduced expression of GLUT1 in 78617GL cells decreases glucose usage, lipid synthesis and proliferation in vitro. A. Immunoblot analysis evaluating the expression of GLUT1, GFP-Luciferase transgene (GFP) and β-actin in lysates from 78617GL cells expressing control shRNA (C) or GLUT1 shRNA (G1). B. Uptake of 3 H-2-deoxyglucose by 78617GL cells expressing control shRNA (shCTRL) or GLUT1 shRNA (shGLUT1) in 15 minutes presented as CPM per µg DNA. C–D. Glucose consumption ( C ) and lactate secretion ( D ). Glucose and lactate concentrations are normalized to the DNA content of the cultures. E. Proliferation is estimated by deteriming the DNA content of cultures at days 0, 1, 2 and 3 post-seeding. F–G. 78617GL cells were grown in soft agar for 3 weeks and colonies are pictured in F and the number of colonies per well is quantified in G . H. Quantification of BrdU positive cells in the outer edge of 12 colonies of each group. I. The concentration of ATP in the two groups of cells (lacking luciferase expression) was determined and normalized to the DNA content of parallel monolayers. J. Lipid synthesis was measured by determining the amount of 14 C in the non-aqueous chloroform fraction of methanol chloroform extracted cell lysates after 24 hour incubation with 14 C-glucose and is normalized to the DNA content of parallel samples. K. qPCR analysis evaluating the expression of the 12 mouse GLUT transporters and SGLT1 in 78617GL cells expressing control shRNA or GLUT1 shRNA normalized to RPL32 expression.

    Article Snippet: Immunoblot analysis Protein was extracted from minced tumor tissue homogenized using a polytron or from plates of cultured cells and immunoblot analysis was performed as previously described using the following antibodies: anti-GLUT1 (Millipore; Billerica, MA or AbCam; Cambridge, MA); anti-cytokeratin 18, anti-GFP and anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA).

    Techniques: Expressing, In Vitro, Luciferase, shRNA, Concentration Assay, Incubation, Real-time Polymerase Chain Reaction

    OTA treatment enhances autophagic flux. ( a ) PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M). At the indicated times after inoculation, the cells were collected, and the expression of p62 and  β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: OTA treatment enhances autophagic flux. ( a ) PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M). At the indicated times after inoculation, the cells were collected, and the expression of p62 and β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Inhibition of autophagy with 3-MA reverses PCV2 replication induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without 3-MA (5 mM). The cells were then assayed for ( a , b ) expression levels of LC3, Cap and  β -actin (loading control), ( c ) PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells, as described in Materials and methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with 3-MA reverses PCV2 replication induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without 3-MA (5 mM). The cells were then assayed for ( a , b ) expression levels of LC3, Cap and β -actin (loading control), ( c ) PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells, as described in Materials and methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Effects of OTA and/or NAC on oxidative stress and autophagy in PCV2-infected PK-15 cells. PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. ( a ) The cells were then incubated with DCFH-DA (10  μ M) at 37 °C for 30 min. The level of ROS was determined by flow cytometry. The level of intracellular ROS, which was indicated by an increase in the fluorescence intensity of the cells, was calculated as the percentage of that of the control cells. ( b ) Representative flourescent staining showing ROS visualized by DCFH-DA fluoreseence (green), and mitochondria labeled by MitoTracker Red CMXRos (red) in PK-15 cells, yellow color (green plus red) indicates the colocalization of ROS and mitochondria. Scale bar: 10  μ m. ( c ) The cells were collected, and the expression levels of LC3, ATG5, Beclin-1 and  β -actin (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. ( d ) PK-15 cells were first transfected with the GFP-LC3 plasmid. After 24 h, the cells were inoculated with PCV2 for 24 h and then incubated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10  μ m. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Effects of OTA and/or NAC on oxidative stress and autophagy in PCV2-infected PK-15 cells. PK-15 cells were inoculated with PCV2 for 24 h and then inculated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. ( a ) The cells were then incubated with DCFH-DA (10  μ M) at 37 °C for 30 min. The level of ROS was determined by flow cytometry. The level of intracellular ROS, which was indicated by an increase in the fluorescence intensity of the cells, was calculated as the percentage of that of the control cells. ( b ) Representative flourescent staining showing ROS visualized by DCFH-DA fluoreseence (green), and mitochondria labeled by MitoTracker Red CMXRos (red) in PK-15 cells, yellow color (green plus red) indicates the colocalization of ROS and mitochondria. Scale bar: 10  μ m. ( c ) The cells were collected, and the expression levels of LC3, ATG5, Beclin-1 and β -actin (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. ( d ) PK-15 cells were first transfected with the GFP-LC3 plasmid. After 24 h, the cells were inoculated with PCV2 for 24 h and then incubated with OTA (0.1  μ M), NAC (5 mM), or OTA and NAC together for an additional 48 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bar: 10  μ m. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Infection, Incubation, Flow Cytometry, Cytometry, Fluorescence, Staining, Labeling, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy

    Inhibition of autophagy with siATG5 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without ATG5 siRNA. The cells were then assayed for the expression levels of ( a ) ATG5 and  β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with siATG5 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without ATG5 siRNA. The cells were then assayed for the expression levels of ( a ) ATG5 and β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Inhibition of autophagy with siBeclin-1 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without Beclin-1 siRNA. Cell were assayed for expression levels of ( a ) Beclin-1 and  β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy with siBeclin-1 reverses the PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with or without Beclin-1 siRNA. Cell were assayed for expression levels of ( a ) Beclin-1 and β -actin (loading control). The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    OTA induces autophagy in PK-15 cells. ( a ) PK-15 cells were inoculated with PCV2 for 24 h, OTA was then added at concentrations of 0.01, 0.1, 1, or 2 μ M, and incubation was continued for an additional 48 h. After collecting the cells, the expression of LC3, ATG5, Beclin-1 and  β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: OTA induces autophagy in PK-15 cells. ( a ) PK-15 cells were inoculated with PCV2 for 24 h, OTA was then added at concentrations of 0.01, 0.1, 1, or 2 μ M, and incubation was continued for an additional 48 h. After collecting the cells, the expression of LC3, ATG5, Beclin-1 and β -actin (loading control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Incubation, Expressing

    Inhibition of autophagy by CQ reverses PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without CQ (5  μ M). Cells were assayed for ( a , b ) expression levels of LC3, Cap and  β -actin (loading control), ( c )PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

    doi: 10.1038/cddis.2017.303

    Figure Lengend Snippet: Inhibition of autophagy by CQ reverses PCV2 replication promotion induced by OTA in PK-15 cells. PCV2-infected cells were incubated with OTA (0.1  μ M) with or without CQ (5  μ M). Cells were assayed for ( a , b ) expression levels of LC3, Cap and β -actin (loading control), ( c )PCV2 viral titers, ( d ) PCV2 viral DNA copies and ( e ) the number of infected cells as described in Materials and Methods. The data are presented as means±S.E. of three independent experiments. Statistical significance compared with the control is indicated by * P

    Article Snippet: Anti-p62, anti-ATG5, anti-Beclin-1, and anti-β -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Infection, Incubation, Expressing

    Gastrodin (GAS) reduced p-tau and Aβ accumulation in the brain of Pb-treated mice. ( A ) Relative density analysis of the Aβ protein in the brain; ( B ) relative density analysis of the p-tau protein bands. The vehicle control is set as 1.0. Total tau or β-actin were probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) reduced p-tau and Aβ accumulation in the brain of Pb-treated mice. ( A ) Relative density analysis of the Aβ protein in the brain; ( B ) relative density analysis of the p-tau protein bands. The vehicle control is set as 1.0. Total tau or β-actin were probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay

    Gastrodin (GAS) increased activated Nrf2 pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Nrf2 pathway in the brain; ( B ) relative density analysis of the Nrf2 protein bands; ( C ) relative density analysis of the HO-1 protein bands; ( D ) relative density analysis of the NQO1 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) increased activated Nrf2 pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Nrf2 pathway in the brain; ( B ) relative density analysis of the Nrf2 protein bands; ( C ) relative density analysis of the HO-1 protein bands; ( D ) relative density analysis of the NQO1 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) activated the Wnt pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Wnt pathway in the brain; ( B ) relative density analysis of the Wnt7a protein bands; ( C ) relative density analysis of the Dkk-1 protein bands; ( D ) relative density analysis of the β-catenin protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) activated the Wnt pathway in the brain of Pb-exposed mice. ( A ) Western blot analysis of the proteins of Wnt pathway in the brain; ( B ) relative density analysis of the Wnt7a protein bands; ( C ) relative density analysis of the Dkk-1 protein bands; ( D ) relative density analysis of the β-catenin protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) inhibited Pb-induced apoptosis in the brain of mice. ( A ) Western blot analysis of the apoptosis-provoking proteins in the brain; ( B ) relative density analysis of the Bcl-2 protein bands; ( C ) relative density analysis of the cytochrome c in cytosol protein bands; ( D ) relative density analysis of the cleaved caspase-3 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) inhibited Pb-induced apoptosis in the brain of mice. ( A ) Western blot analysis of the apoptosis-provoking proteins in the brain; ( B ) relative density analysis of the Bcl-2 protein bands; ( C ) relative density analysis of the cytochrome c in cytosol protein bands; ( D ) relative density analysis of the cleaved caspase-3 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gastrodin (GAS) alleviated Pb-induced synaptic dysfunction in the brain of mice. ( A ) Relative density analysis of the BDNF protein bands; ( B ) relative density analysis of the NR2A protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) alleviated Pb-induced synaptic dysfunction in the brain of mice. ( A ) Relative density analysis of the BDNF protein bands; ( B ) relative density analysis of the NR2A protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay

    Gastrodin (GAS) inhibited Pb-induced inflammation in the brain of mice. ( A ) Western blot analysis of the apoptosis-related proteins in the brain; ( B ) relative density analysis of the NF-κB protein bands; ( C ) relative density analysis of the TNF-α protein bands; ( D ) relative density analysis of the COX-2 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Journal: Nutrients

    Article Title: Effects of Gastrodin against Lead-Induced Brain Injury in Mice Associated with the Wnt/Nrf2 Pathway

    doi: 10.3390/nu12061805

    Figure Lengend Snippet: Gastrodin (GAS) inhibited Pb-induced inflammation in the brain of mice. ( A ) Western blot analysis of the apoptosis-related proteins in the brain; ( B ) relative density analysis of the NF-κB protein bands; ( C ) relative density analysis of the TNF-α protein bands; ( D ) relative density analysis of the COX-2 protein bands. β-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Data are expressed as mean ± S.E.M. and representative of five independent experiments (individual animals). ## p

    Article Snippet: The p-tau, Aβ, Wnt7a, β-catenin, NR2A, BDNF, Nrf2, HO-1, NQO1, TNF-α, COX-2, Bcl-2, cytochrome C, cleaved caspase-3 and β-actin antibodies were supplied by Santa Cruz Biotechnology (CA, USA) and Abcam (Cambridge, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P

    Journal: Frontiers in Physiology

    Article Title: Exercise and Omentin: Their Role in the Crosstalk Between Muscle and Adipose Tissues in Type 2 Diabetes Mellitus Rat Models

    doi: 10.3389/fphys.2018.01881

    Figure Lengend Snippet: Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P

    Article Snippet: Protein concentrations were normalized by using β-actin diluted 1:2,000 (Cell Signaling Technology, Beverly, MA, United States) in the visceral fat, or GAPDH diluted 1:10,000 (Abcam) in muscle. β-actin was detected with mouse peroxidase (HRP) – conjugated with a second antibody (Cell Signaling) diluted 1:3,000 in TBS-T, and GAPDH using antirabbit antibody (Cell Signaling), incubated and agitated for 1 h at room temperature.

    Techniques: Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

    Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing

    Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Rescue of KIF3A expression in Kif3a -/- MEFs does not attenuate RHOA activation mediated by purmorphamine. A . GLI1 induction and KIF3A expression in WT MEFs (WT) vs. Kif3a -/- MEFs infected with AdV-Kif3a at MOIs of 10 and 80. p38 was used as loading control (n = 3). B . Staining of primary cilia in WT MEFs, Kif3a -/- MEFs, or Kif3a -/- MEFs infected with either control (empty) AdV or AdV-Kif3a (MOI = 10). Cells were stained with anti-acetylated α-tubulin and anti-Alexa 488 to visualize the axoneme of each primary cilium. Quantification of the percentage of cilia-positive cells (mean ± S.E.M; n = 4) C . RHOA activation in Kif3a -/- MEFs infected with control AdV or AdV-Kif3a at MOI = 10 and stimulated with 5 μM PUR for the indicated times (β-actin was used as loading control because total RHOA was obscured by a cross-reactive protein that appears as a consequence of AdV infection). D . Densitometric quantification of RHOA-GTP/ β-actin increase in response to 5 μM PUR at the indicated times. The ratios were expressed as fold change compared to t = 0. * p

    Journal: PLoS ONE

    Article Title: Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia

    doi: 10.1371/journal.pone.0203170

    Figure Lengend Snippet: Rescue of KIF3A expression in Kif3a -/- MEFs does not attenuate RHOA activation mediated by purmorphamine. A . GLI1 induction and KIF3A expression in WT MEFs (WT) vs. Kif3a -/- MEFs infected with AdV-Kif3a at MOIs of 10 and 80. p38 was used as loading control (n = 3). B . Staining of primary cilia in WT MEFs, Kif3a -/- MEFs, or Kif3a -/- MEFs infected with either control (empty) AdV or AdV-Kif3a (MOI = 10). Cells were stained with anti-acetylated α-tubulin and anti-Alexa 488 to visualize the axoneme of each primary cilium. Quantification of the percentage of cilia-positive cells (mean ± S.E.M; n = 4) C . RHOA activation in Kif3a -/- MEFs infected with control AdV or AdV-Kif3a at MOI = 10 and stimulated with 5 μM PUR for the indicated times (β-actin was used as loading control because total RHOA was obscured by a cross-reactive protein that appears as a consequence of AdV infection). D . Densitometric quantification of RHOA-GTP/ β-actin increase in response to 5 μM PUR at the indicated times. The ratios were expressed as fold change compared to t = 0. * p

    Article Snippet: The β-actin antibody (Cat#A1978 clone AC-15) was purchased from Sigma-Aldrich and used at 1:10000–1:40000 dilution.

    Techniques: Expressing, Activation Assay, Infection, Staining

    CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. β-actin was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P

    Journal: Nature Communications

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice

    doi: 10.1038/s41467-018-03008-2

    Figure Lengend Snippet: CCP6 or CCP1 deficiency promotes cell reprogramming. a Ccp1 - or Ccp6 - deficient MEFs were lyzed for immunoblotting. β-actin was used as a loading control. b WT or CCPs-deficient MEFs were infected by OSKM factors containing retrovirus and cultured in ESC media for 3 weeks. Alkaline phosphatase (AP)-positive colony numbers per 10 4 cells were calculated and shown as means ± S.D. **, P

    Article Snippet: Antibodies against Flag-tag (M2, F3165), β-actin (A-5316), His-tag (H1029), glutamylated tubulin (B3), and GFP-tag (G-1544) were from Sigma-Aldrich (St. Louis, USA).

    Techniques: Infection, Cell Culture

    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.

    Journal: Journal of Toxicologic Pathology

    Article Title: Spontaneous pulmonary co-metastasis of hepatoblastoma arising within a hepatocellular carcinoma in an aged C57BL/6J mouse

    doi: 10.1293/tox.2017-0067

    Figure Lengend Snippet: Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.

    Article Snippet: Endogenous peroxidase was blocked by incubating sections in 3% H2 O2 for 15 min. For antigen retrieval, sections were immersed in citrate buffer (pH 6.0), heated, and cooled at room temperature. β-catenin (rabbit monoclonal, clone 6B3, dilution 1:150, Cell Signaling) specific antibody was applied for 1.5 hours at room temperature.

    Techniques: Immunohistochemistry, Staining

    Model of single β-catenin mutation and interaction with E-cadherin in the small intestine and colon A, B Model of a single activating β-catenin mutation in the murine small intestine and the colon. In contrast to the small intestine (A), the increased levels of E-cadherin in the colon complexed with mutant β-catenin prevent its accumulation in the nucleus (B).

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Model of single β-catenin mutation and interaction with E-cadherin in the small intestine and colon A, B Model of a single activating β-catenin mutation in the murine small intestine and the colon. In contrast to the small intestine (A), the increased levels of E-cadherin in the colon complexed with mutant β-catenin prevent its accumulation in the nucleus (B).

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mutagenesis

    Human cancers, characterised by β-catenin mutation, are associated with reduction in E-cadherin levels Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann–Whitney U -test, P = 0.0009051. White scale bar, 50 μm. Correlation of several Wnt target genes with the expression of E-cadherin ( CDH1 ) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Human cancers, characterised by β-catenin mutation, are associated with reduction in E-cadherin levels Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann–Whitney U -test, P = 0.0009051. White scale bar, 50 μm. Correlation of several Wnt target genes with the expression of E-cadherin ( CDH1 ) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mutagenesis, Proximity Ligation Assay, Microarray, Single-particle Tracking, Staining, MANN-WHITNEY, Expressing

    Rapid colonic phenotype in VilCre ER Catnb lox(ex3)/lox(ex3) and Apc fl/fl mice, but not in VilCreER Catnb lox(ex3)/ + mice Wild-type mice show very little nuclear β-catenin, and the expression of Sox9 is restricted to the bottom of the crypt. VilCreER Catnb lox(ex3)/ + show a similar phenotype. In contrast, VilCre ER Catnb lox(ex3)/lox(ex3) and VilCre ER Apc fl/fl mice show increased nuclear β-catenin and high expression of Sox9. Scale bar, 100 μm. Mice were sampled at day 4 after induction.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Rapid colonic phenotype in VilCre ER Catnb lox(ex3)/lox(ex3) and Apc fl/fl mice, but not in VilCreER Catnb lox(ex3)/ + mice Wild-type mice show very little nuclear β-catenin, and the expression of Sox9 is restricted to the bottom of the crypt. VilCreER Catnb lox(ex3)/ + show a similar phenotype. In contrast, VilCre ER Catnb lox(ex3)/lox(ex3) and VilCre ER Apc fl/fl mice show increased nuclear β-catenin and high expression of Sox9. Scale bar, 100 μm. Mice were sampled at day 4 after induction.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mouse Assay, Expressing

    Single copy activation of β-catenin only slowly transforms the intestine Only activation of both alleles of β-catenin led to hyperproliferation in the small intestine. Proliferation of wild-type (WT), AhCre ER Catnb lox(ex3)/ + and AhCre ER Catnb lox(ex3)/lox(ex3) mice 5 days after induction was scored by counting the number of BrdU-positive cells/half-crypt. N ≥ 3 per group, at least 25 crypts per mouse were scored, P -value of one-sided Mann–Whitney U -test. Activation of one of copy β-catenin in an aged AhCre ER Catnb lox(ex3)/ + at day 25 post-induction with no phenotype in the colon. For comparison to a WT colon, see Appendix Fig S3 . Scale bar, 100 μm. Activation of one copy of β-catenin in VilCreER Catnb lox(ex3)/ + leads to the same crypt-progenitor cell phenotype (red bar) with similar kinetics as observed in AhCre ER Catnb lox(ex3)/ + mice. Scale bar, 100 μm.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Single copy activation of β-catenin only slowly transforms the intestine Only activation of both alleles of β-catenin led to hyperproliferation in the small intestine. Proliferation of wild-type (WT), AhCre ER Catnb lox(ex3)/ + and AhCre ER Catnb lox(ex3)/lox(ex3) mice 5 days after induction was scored by counting the number of BrdU-positive cells/half-crypt. N ≥ 3 per group, at least 25 crypts per mouse were scored, P -value of one-sided Mann–Whitney U -test. Activation of one of copy β-catenin in an aged AhCre ER Catnb lox(ex3)/ + at day 25 post-induction with no phenotype in the colon. For comparison to a WT colon, see Appendix Fig S3 . Scale bar, 100 μm. Activation of one copy of β-catenin in VilCreER Catnb lox(ex3)/ + leads to the same crypt-progenitor cell phenotype (red bar) with similar kinetics as observed in AhCre ER Catnb lox(ex3)/ + mice. Scale bar, 100 μm.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Activation Assay, Mouse Assay, MANN-WHITNEY

    Apc loss, GSK3 loss and homozygous mutation of β-catenin are sufficient to induce a rapid crypt-progenitor phenotype, but not a single β-catenin mutation Wild-type mice have small defined crypts in the small intestine with little nuclear β-catenin at the bottom of the crypt. The small intestine of AhCre ER APC fl/fl , AhCre ER Catnb lox(ex3)/lox(ex3) (day 5) or AhCre ER Gsk3alpha fl/fl beta fl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclear β-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm. A heterozygous activation of β-catenin ( AhCre ER Catnb lox(ex3)/ + ) shows no increase in crypt size or nuclear β-catenin at days 5–10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm. Culture of small intestinal crypts of WT and VilCre ER Apc fl/fl (or AhCre ER Apc fl/fl ) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc -deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm. At day 5 post-induction, only crypts from AhCre ER Catnb lox(ex3)/lox(ex3) but not AhCre ER Catnb lox(ex3)/ + survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCre ER Catnb lox(ex3)/ + compared to spheres from Catnb lox(ex3)/lox(ex3) crypts in the first week of culture. Black scale bar, 50 μm. Quantification of organoids/spheres at day 10 post-induction ( N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Apc loss, GSK3 loss and homozygous mutation of β-catenin are sufficient to induce a rapid crypt-progenitor phenotype, but not a single β-catenin mutation Wild-type mice have small defined crypts in the small intestine with little nuclear β-catenin at the bottom of the crypt. The small intestine of AhCre ER APC fl/fl , AhCre ER Catnb lox(ex3)/lox(ex3) (day 5) or AhCre ER Gsk3alpha fl/fl beta fl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclear β-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm. A heterozygous activation of β-catenin ( AhCre ER Catnb lox(ex3)/ + ) shows no increase in crypt size or nuclear β-catenin at days 5–10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm. Culture of small intestinal crypts of WT and VilCre ER Apc fl/fl (or AhCre ER Apc fl/fl ) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc -deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm. At day 5 post-induction, only crypts from AhCre ER Catnb lox(ex3)/lox(ex3) but not AhCre ER Catnb lox(ex3)/ + survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCre ER Catnb lox(ex3)/ + compared to spheres from Catnb lox(ex3)/lox(ex3) crypts in the first week of culture. Black scale bar, 50 μm. Quantification of organoids/spheres at day 10 post-induction ( N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mutagenesis, Mouse Assay, Activation Assay

    Haploinsufficiency for E-cadherin in the presence of single allele β-catenin mutation leads to Wnt deregulation AhCre ER Catnb lox(ex3)/ + compared to AhCre ER Catnb lox(ex3)/ + Cdh1 fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCre ER Catnb lox(ex3)/ + Cdh1 fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU). Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCre ER Cdh1 fl/ + , AhCre ER Catnb lox(ex3)/ + and AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice ( P = 0.028, one-sided Mann–Whitney U -test). In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCre ER Catnb lox(ex3)/ + AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + , day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann–Whitney U -test. Survival of Lgr5Cre ER Catnb lox(ex3)/ + and Lgr5Cre ER Catnb lox(ex3)/ + Cdh1 fl/ + shows significant acceleration ( P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-catenin IHC in contrast to Lgr5Cre ER Catnb lox(ex3)/ + mice.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Haploinsufficiency for E-cadherin in the presence of single allele β-catenin mutation leads to Wnt deregulation AhCre ER Catnb lox(ex3)/ + compared to AhCre ER Catnb lox(ex3)/ + Cdh1 fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCre ER Catnb lox(ex3)/ + Cdh1 fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU). Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCre ER Cdh1 fl/ + , AhCre ER Catnb lox(ex3)/ + and AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice ( P = 0.028, one-sided Mann–Whitney U -test). In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCre ER Catnb lox(ex3)/ + AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + , day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann–Whitney U -test. Survival of Lgr5Cre ER Catnb lox(ex3)/ + and Lgr5Cre ER Catnb lox(ex3)/ + Cdh1 fl/ + shows significant acceleration ( P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-catenin IHC in contrast to Lgr5Cre ER Catnb lox(ex3)/ + mice.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mutagenesis, Mouse Assay, MANN-WHITNEY, In Vitro, Immunohistochemistry

    Haploinsufficiency for E-cadherin lowers the threshold for Wnt activation of β-catenin mutation AhCre ER Cdh1 fl/ + at day 5 post-induction shows no intestinal phenotype. AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice show increased proliferation (BrdU, red line) and accumulation of β-catenin and Sox9 in cells higher up the crypt-villus axis (arrows). Scale bar, 100 μm. Increased proliferation as scored by BrdU + cells per half-crypt. N ≥ 3, statistics: one-sided Mann–Whitney U -test.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Haploinsufficiency for E-cadherin lowers the threshold for Wnt activation of β-catenin mutation AhCre ER Cdh1 fl/ + at day 5 post-induction shows no intestinal phenotype. AhCre ER Catnb lox(ex3)/ + Cdh1 fl/ + mice show increased proliferation (BrdU, red line) and accumulation of β-catenin and Sox9 in cells higher up the crypt-villus axis (arrows). Scale bar, 100 μm. Increased proliferation as scored by BrdU + cells per half-crypt. N ≥ 3, statistics: one-sided Mann–Whitney U -test.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Activation Assay, Mutagenesis, Mouse Assay, MANN-WHITNEY

    E-cadherin saturates with mutant β-catenin over time Immunoprecipitation (IP) of E-cadherin from VilCre ER Catnb lox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments ( N = 3 mice for each genotype and time point); error bars represent s.e.m. IP of E-cadherin from VilCre ER Catnb lox(ex3)/ + mice at day 4 and day 8. Graphs show average of experiments ( N = 3 mice for each genotype and time point); error bars represent s.e.m.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: E-cadherin saturates with mutant β-catenin over time Immunoprecipitation (IP) of E-cadherin from VilCre ER Catnb lox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments ( N = 3 mice for each genotype and time point); error bars represent s.e.m. IP of E-cadherin from VilCre ER Catnb lox(ex3)/ + mice at day 4 and day 8. Graphs show average of experiments ( N = 3 mice for each genotype and time point); error bars represent s.e.m.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mutagenesis, Immunoprecipitation, Mouse Assay

    Increased E-cadherin:β-catenin levels in colonic crypts of wild-type mice Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm. Western blot of purified crypts from the small intestine and colon ( N = 4 mice). qRT–PCR of whole tissue from small intestine and colon. Expression of mRNA (2 (−d C t) ) calculated relative to GAPDH ( N = 3 mice). Statistics: one-sided Mann–Whitney U -test. Proximity ligation assay for E-cadherin and β-catenin. For each mouse ( N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann–Whitney U -test; white scale bar, 50 μm.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Increased E-cadherin:β-catenin levels in colonic crypts of wild-type mice Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm. Western blot of purified crypts from the small intestine and colon ( N = 4 mice). qRT–PCR of whole tissue from small intestine and colon. Expression of mRNA (2 (−d C t) ) calculated relative to GAPDH ( N = 3 mice). Statistics: one-sided Mann–Whitney U -test. Proximity ligation assay for E-cadherin and β-catenin. For each mouse ( N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann–Whitney U -test; white scale bar, 50 μm.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mouse Assay, Staining, Western Blot, Purification, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Proximity Ligation Assay

    Activation of one allele of β-catenin in Lgr5-positive stem cells does not result in colonic lesions Table shows that only homozygous activation of β-catenin ( Lgr5Cre ER Catnb lox(ex3)/lox(ex3) ) or loss of Apc ( Lgr5Cre ER Apc fl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin ( Lgr5Cre ER Catnb lox(ex3)/ + ) was mutated (chi-squared test, P

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Activation of one allele of β-catenin in Lgr5-positive stem cells does not result in colonic lesions Table shows that only homozygous activation of β-catenin ( Lgr5Cre ER Catnb lox(ex3)/lox(ex3) ) or loss of Apc ( Lgr5Cre ER Apc fl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin ( Lgr5Cre ER Catnb lox(ex3)/ + ) was mutated (chi-squared test, P

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Activation Assay

    Only deletion of both Gsk3alpha and Gsk3beta leads to crypt-progenitor-cell phenotype Small Intestine of mice at day 6 after induction. Loss of Gsk3alpha and Gsk3beta ( AhCre ER Gsk3alpha fl/fl Gsk3beta fl/fl ) leads to accumulation of nuclear β-catenin and upregulation of cMyc (arrows). The crypt-progenitor cell phenotype (red bar) is characterised by increased proliferation (BrdU) and perturbed differentiation/localisation of goblet and Paneth cells (Alcian Blue, Lysozyme respectively, arrows). Scale bar, 100 μm. Table shows cohort of AhCre Gsk3alpha Gsk3beta mice aged until signs of intestinal tumour burden, genotypes as indicated. Note, mice homozygous for Gsk3beta deletion, or with only one copy of GSK3alpha and GSK3beta , did not develop intestinal tumours. An adenoma from an aged mouse deficient for 3 alleles of Gsk3alpha and Gsk3beta (AhCre GSK3alpha fl/fl beta fl/ + ) showing that it developed after loss of the remaining GKS3beta allele. Scale bar, 100 μm.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Only deletion of both Gsk3alpha and Gsk3beta leads to crypt-progenitor-cell phenotype Small Intestine of mice at day 6 after induction. Loss of Gsk3alpha and Gsk3beta ( AhCre ER Gsk3alpha fl/fl Gsk3beta fl/fl ) leads to accumulation of nuclear β-catenin and upregulation of cMyc (arrows). The crypt-progenitor cell phenotype (red bar) is characterised by increased proliferation (BrdU) and perturbed differentiation/localisation of goblet and Paneth cells (Alcian Blue, Lysozyme respectively, arrows). Scale bar, 100 μm. Table shows cohort of AhCre Gsk3alpha Gsk3beta mice aged until signs of intestinal tumour burden, genotypes as indicated. Note, mice homozygous for Gsk3beta deletion, or with only one copy of GSK3alpha and GSK3beta , did not develop intestinal tumours. An adenoma from an aged mouse deficient for 3 alleles of Gsk3alpha and Gsk3beta (AhCre GSK3alpha fl/fl beta fl/ + ) showing that it developed after loss of the remaining GKS3beta allele. Scale bar, 100 μm.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Mouse Assay

    Complete loss of Gsk3 in Lgr5-positive stem cells leads to tumour formation in the small intestine and the colon Immunofluorescence analysis shows intestinal lesions with accumulation of β-catenin and SOX9 in the small intestine and the colon, 25 days after induction. Scale bar, 50 μm.

    Journal: The EMBO Journal

    Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations

    doi: 10.15252/embj.201591739

    Figure Lengend Snippet: Complete loss of Gsk3 in Lgr5-positive stem cells leads to tumour formation in the small intestine and the colon Immunofluorescence analysis shows intestinal lesions with accumulation of β-catenin and SOX9 in the small intestine and the colon, 25 days after induction. Scale bar, 50 μm.

    Article Snippet: Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin (1/1,000 BD Biosciences, #610154).

    Techniques: Immunofluorescence

    Inactivation of β-catenin results in aberrant expression of SOX9 in a subset of airway epithelial cells Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) and control β-catenin fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for SOX9, SOX2 (A) and β-catenin (B). β-catenin-deficient embryos show aberrant expression of SOX9 in airway epithelium, which was identified by SOX2 (A–B). Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. Abbreviations: Br, bronchi; Cart, cartilage. Magnifications: main images, ×400; inserts, ×800.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Inactivation of β-catenin results in aberrant expression of SOX9 in a subset of airway epithelial cells Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) and control β-catenin fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for SOX9, SOX2 (A) and β-catenin (B). β-catenin-deficient embryos show aberrant expression of SOX9 in airway epithelium, which was identified by SOX2 (A–B). Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. Abbreviations: Br, bronchi; Cart, cartilage. Magnifications: main images, ×400; inserts, ×800.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Expressing, Mouse Assay

    Inactivation of β-catenin causes accumulation of SOX9-positive basal cells in developing airways of E16.5 embryos Paraffin sections were prepared from E16.5 embryos. Dox was given to pregnant mice from E0.5 to E16.5. Slides were immunostained for SOX9 (green) and p63 (red). DAPI was used to label cell nuclei (blue). Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) lungs show accumulation of SOX9-positive basal cells in developing airways (shown by dotted lines). Magnifications: left and middle panels, ×400; right panels, ×800.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Inactivation of β-catenin causes accumulation of SOX9-positive basal cells in developing airways of E16.5 embryos Paraffin sections were prepared from E16.5 embryos. Dox was given to pregnant mice from E0.5 to E16.5. Slides were immunostained for SOX9 (green) and p63 (red). DAPI was used to label cell nuclei (blue). Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) lungs show accumulation of SOX9-positive basal cells in developing airways (shown by dotted lines). Magnifications: left and middle panels, ×400; right panels, ×800.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Mouse Assay

    Deletion of β-catenin induces SOX9 in conducting airways and increases the number of basal cells at E12.5 Paraffin sections were prepared from E12.5 embryos. Dox was given to pregnant mice from E0.5 to E12.5. Lung sections were immunostained for SOX9, SOX2, β-catenin and p63. DAPI was used to label cell nuclei. Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) lungs show accumulation of SOX9-positive basal cells in conducting airways (dotted lines). Abbreviations: Br, bronchi; Cart, cartilage. Magnification is ×400.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Deletion of β-catenin induces SOX9 in conducting airways and increases the number of basal cells at E12.5 Paraffin sections were prepared from E12.5 embryos. Dox was given to pregnant mice from E0.5 to E12.5. Lung sections were immunostained for SOX9, SOX2, β-catenin and p63. DAPI was used to label cell nuclei. Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) lungs show accumulation of SOX9-positive basal cells in conducting airways (dotted lines). Abbreviations: Br, bronchi; Cart, cartilage. Magnification is ×400.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Mouse Assay

    SOX9 does not affect expression of β-catenin in the developing respiratory epithelium Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/Sox9 fl/fl ( SPC/Sox9 −/− ) and control Sox9 fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for β-catenin, SOX9 and Cyclin D1. DAPI was used to label cell nuclei. SOX9 and β-catenin are co-expressed in distal respiratory epithelium of control lungs (A). Deletion of SOX9 does not affect expression of β-catenin (A) or Cyclin D1. Magnification is ×400.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: SOX9 does not affect expression of β-catenin in the developing respiratory epithelium Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/Sox9 fl/fl ( SPC/Sox9 −/− ) and control Sox9 fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for β-catenin, SOX9 and Cyclin D1. DAPI was used to label cell nuclei. SOX9 and β-catenin are co-expressed in distal respiratory epithelium of control lungs (A). Deletion of SOX9 does not affect expression of β-catenin (A) or Cyclin D1. Magnification is ×400.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Expressing, Mouse Assay

    SOX9 replaces SOX2 in airway epithelium of SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) mice Lung sections from Dox-treated SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) and control β-catenin fl/fl E14.5 embryos were stained for SOX9, SOX2 and β-catenin. Ectopic expression of SOX9 and loss of SOX2 is observed in airway epithelium of SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) mice (A). Triple immunostaining for SOX9, SOX2 and β-catenin revealed that the highest expression of β-catenin is associated with the loss of SOX2 and aberrant expression of SOX9 in airway epithelial cells (B, arrowheads). DAPI was used to label cell nuclei. Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. Abbreviations: Br, bronchi. Magnification is ×400.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: SOX9 replaces SOX2 in airway epithelium of SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) mice Lung sections from Dox-treated SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) and control β-catenin fl/fl E14.5 embryos were stained for SOX9, SOX2 and β-catenin. Ectopic expression of SOX9 and loss of SOX2 is observed in airway epithelium of SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) mice (A). Triple immunostaining for SOX9, SOX2 and β-catenin revealed that the highest expression of β-catenin is associated with the loss of SOX2 and aberrant expression of SOX9 in airway epithelial cells (B, arrowheads). DAPI was used to label cell nuclei. Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. Abbreviations: Br, bronchi. Magnification is ×400.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Mouse Assay, Staining, Expressing, Triple Immunostaining

    Inactivation of β-catenin inhibits SOX9 in the peripheral lung Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5). Slides were stained with Hematoxilin and Eosin (H E) or used for immunostaining with β-catenin, SOX9 and TTF-1 (Nkx2.1) antibodies. Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SP-C/β-catenin −/− ) embryos showed enlarged epithelial cysts in peripheral lung regions (A). Loss of β-catenin did not affect TTF-1 (C) but decreased SOX9 and Cyclin D1 in cystic lung epithelium (B, arrowheads). DAPI was used to label cell nuclei. Yellow arrows show distal epithelial tubules. White arrows show epithelial cysts. Abbreviations: Br, bronchi; Cyst, peripheral epithelial cysts; dt, distal tubules. Magnifications: ×100 (A); ×200 (right panel in A); ×400 (B–C); and ×800 (inserts).

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Inactivation of β-catenin inhibits SOX9 in the peripheral lung Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5). Slides were stained with Hematoxilin and Eosin (H E) or used for immunostaining with β-catenin, SOX9 and TTF-1 (Nkx2.1) antibodies. Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SP-C/β-catenin −/− ) embryos showed enlarged epithelial cysts in peripheral lung regions (A). Loss of β-catenin did not affect TTF-1 (C) but decreased SOX9 and Cyclin D1 in cystic lung epithelium (B, arrowheads). DAPI was used to label cell nuclei. Yellow arrows show distal epithelial tubules. White arrows show epithelial cysts. Abbreviations: Br, bronchi; Cyst, peripheral epithelial cysts; dt, distal tubules. Magnifications: ×100 (A); ×200 (right panel in A); ×400 (B–C); and ×800 (inserts).

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Mouse Assay, Staining, Immunostaining

    Deletion of β-catenin in the lung epithelium does not affect SOX2 expression Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice from E0.5 to E14.5. Slides were immunostained for SOX9 (green), SOX2 (cyan) and β-catenin (red). Deletion of β-catenin does not influence expression of SOX2 in airway epithelial cells. Magnification is ×400.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Deletion of β-catenin in the lung epithelium does not affect SOX2 expression Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice from E0.5 to E14.5. Slides were immunostained for SOX9 (green), SOX2 (cyan) and β-catenin (red). Deletion of β-catenin does not influence expression of SOX2 in airway epithelial cells. Magnification is ×400.

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Expressing, Mouse Assay

    Inactivation of β-catenin increases the numbers of basal cells and causes accumulation of SOX9-positive basal cells in E14.5 lungs A–B, SOX9 co-localizes with basal cell markers in β-catenin-deficient airway epithelium. Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) and control β-catenin fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for SOX9, p63 and T1α. Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. β-Catenin-deficient embryos show co-localization of SOX9 with p63 (A, arrows). Nuclear SOX9 staining is observed in T1α-positive basal cells (B, inserts). C, Inactivation of β-catenin increases the number of basal cells in airway epithelium. The numbers of p63-positive and p63/SOX9 double-positive cells were counted in 20 airway regions selected randomly (n=5 embryos in each group). * indicates statistically significant changes (p

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Inactivation of β-catenin increases the numbers of basal cells and causes accumulation of SOX9-positive basal cells in E14.5 lungs A–B, SOX9 co-localizes with basal cell markers in β-catenin-deficient airway epithelium. Paraffin sections were prepared from E14.5 lungs obtained from Dox-treated SPC-rtTA/tetO-Cre/β-catenin fl/fl ( SPC/β-catenin −/− ) and control β-catenin fl/fl embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5) until the animal harvest. Slides were immunostained for SOX9, p63 and T1α. Dotted lines indicate boundaries of epithelial layers in pulmonary bronchi. β-Catenin-deficient embryos show co-localization of SOX9 with p63 (A, arrows). Nuclear SOX9 staining is observed in T1α-positive basal cells (B, inserts). C, Inactivation of β-catenin increases the number of basal cells in airway epithelium. The numbers of p63-positive and p63/SOX9 double-positive cells were counted in 20 airway regions selected randomly (n=5 embryos in each group). * indicates statistically significant changes (p

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Mouse Assay, Staining

    Activation of β-catenin in respiratory epithelium induces SOX9 Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5). Slides were stained with Hematoxilin and Eosin (H E) or used for immunostaining with β-catenin and SOX9 antibodies. SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) lungs show abnormal cyst-like structures in the lung tissue (A). Activated β-catenin increases SOX9 staining in cystic epithelium (B, arrowheads). DAPI was used to label cell nuclei. Abbreviations: Br, bronchi. Magnifications: ×100 (A) and ×400 (B).

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: β-Catenin and Kras/Foxm1 Signaling Pathway Are Critical to Restrict Sox9 in Basal Cells During Pulmonary Branching Morphogenesis

    doi: 10.1002/dvdy.24393

    Figure Lengend Snippet: Activation of β-catenin in respiratory epithelium induces SOX9 Paraffin sections were prepared from E14.5 embryos. Dox was given to pregnant mice after identification of a vaginal plug (E0.5). Slides were stained with Hematoxilin and Eosin (H E) or used for immunostaining with β-catenin and SOX9 antibodies. SPC-rtTA/TetO-Cre/β-catenin +/Δ(ex3) lungs show abnormal cyst-like structures in the lung tissue (A). Activated β-catenin increases SOX9 staining in cystic epithelium (B, arrowheads). DAPI was used to label cell nuclei. Abbreviations: Br, bronchi. Magnifications: ×100 (A) and ×400 (B).

    Article Snippet: The following antibodies were used for immunostaining: SOX9 (AB5535, Millipore and AF3075, R & D Systems), SOX2 , β-catenin (sc-1496, Santa Cruz Biotechnology), TTF1 (Nkx2.1) (R1231, Seven Hills Bioreagents), T1α ( ); p63 (sc71827, Santa Cruz Biotechnology); cyclin D1 (ab7958, abcam); cytokeratin 5 (a gift from J. Whitsett, Cincinnati Children’s Hospital) and β-galactosidase ( ).

    Techniques: Activation Assay, Mouse Assay, Staining, Immunostaining

    Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S and Tom20 C21S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S and Tom20 C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO 4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO 4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO 4 -induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO 4 -induced cytochrome c release detected in the cytosol fraction was diminished ( d ), the cleavage of caspase-3 and -9 was attenuated ( e ), the cell viability was rescued ( f ), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished ( g ). h Effects of the mutants Tom20 C13S and Tom20 C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO 4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S and Tom20 C21S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S and Tom20 C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO 4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO 4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO 4 -induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO 4 -induced cytochrome c release detected in the cytosol fraction was diminished ( d ), the cleavage of caspase-3 and -9 was attenuated ( e ), the cell viability was rescued ( f ), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished ( g ). h Effects of the mutants Tom20 C13S and Tom20 C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO 4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques: Translocation Assay, Transfection, Confocal Microscopy

    Iron acts as a sensitizer for different drugs and induces pyroptosis in melanoma cells. Melanoma A375 cells were treated with SSZ (sulfasalazine, 125 μM) with or without FeSO 4 (100 μM) for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a FeSO 4 acts as a sensitizer for ROS generation, GSDME cleavage, LDH release, and pyroptosis in the presence of SSZ. b , c Extensive treatment with SSZ/FeSO 4 at the indicated times induced Tom20 oxidation and accumulation ( b ) and GSDME cleavage ( c ). d – g Separate knockdown of Tom20, Bax, caspase-3 or GSDME blocked SSZ/FeSO 4 -induced pyroptosis, GSDME cleavage, and LDH release as indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Iron acts as a sensitizer for different drugs and induces pyroptosis in melanoma cells. Melanoma A375 cells were treated with SSZ (sulfasalazine, 125 μM) with or without FeSO 4 (100 μM) for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a FeSO 4 acts as a sensitizer for ROS generation, GSDME cleavage, LDH release, and pyroptosis in the presence of SSZ. b , c Extensive treatment with SSZ/FeSO 4 at the indicated times induced Tom20 oxidation and accumulation ( b ) and GSDME cleavage ( c ). d – g Separate knockdown of Tom20, Bax, caspase-3 or GSDME blocked SSZ/FeSO 4 -induced pyroptosis, GSDME cleavage, and LDH release as indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques:

    Iron activates ROS to promote the pyroptotic death of melanoma cells. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM), for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell viability, unless specifically defined. a The addition of FeSO 4 to CCCP induced cell death, LDH release, and pyroptosis (pyroptotic cells are indicated by red arrows). b Knockdown of FTL or FTH1 enhanced LDH release and cell death in response to CCCP/FeSO 4 stimulation. KD: knockdown. c Knockdown of GSDME diminished FeSO 4 -induced pyroptosis and LDH release in the presence of CCCP. d Cleavage of GSDME was observed in response to CCCP/FeSO 4 stimulation. e The effects of NAC or GSH on CCCP/FeSO 4 -induced ROS elevation and cell death. f GSH blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. g Knockdown of FTL or FTH1 enhanced CCCP/FeSO 4 -induced LDH release and GSDME cleavage. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Iron activates ROS to promote the pyroptotic death of melanoma cells. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM), for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell viability, unless specifically defined. a The addition of FeSO 4 to CCCP induced cell death, LDH release, and pyroptosis (pyroptotic cells are indicated by red arrows). b Knockdown of FTL or FTH1 enhanced LDH release and cell death in response to CCCP/FeSO 4 stimulation. KD: knockdown. c Knockdown of GSDME diminished FeSO 4 -induced pyroptosis and LDH release in the presence of CCCP. d Cleavage of GSDME was observed in response to CCCP/FeSO 4 stimulation. e The effects of NAC or GSH on CCCP/FeSO 4 -induced ROS elevation and cell death. f GSH blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. g Knockdown of FTL or FTH1 enhanced CCCP/FeSO 4 -induced LDH release and GSDME cleavage. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques:

    Mitochondrial pathway with activation of caspase-3 is involved in pyroptosis induced by iron. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a Mitochondrial depletion blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. Mito: mitochondria. b CCCP/FeSO 4 induced mitochondrial accumulation, but NAC and GSH attenuated this accumulation. c CCCP/FeSO 4 induced cytochrome c release from mitochondria to cytosol as detected in the cytosol fraction (left) or by confocal microscopy (right). Cyto C: cytochrome c. d , e NAC or GSH abolished CCCP/FeSO 4 -induced cytochrome c release ( d ) and cleavage of caspase-3 and -9 ( e ). CASP: caspase. f Knockdown of FTL or FTH1 enhanced the CCCP/FeSO 4 -induced cleavage of caspase-3 and -9. g , h Knockdown of either caspase-3 or -9 abolished the CCCP/FeSO 4 -induced GSDME cleavage ( g ), pyroptosis and LDH release ( h ). Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Mitochondrial pathway with activation of caspase-3 is involved in pyroptosis induced by iron. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a Mitochondrial depletion blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. Mito: mitochondria. b CCCP/FeSO 4 induced mitochondrial accumulation, but NAC and GSH attenuated this accumulation. c CCCP/FeSO 4 induced cytochrome c release from mitochondria to cytosol as detected in the cytosol fraction (left) or by confocal microscopy (right). Cyto C: cytochrome c. d , e NAC or GSH abolished CCCP/FeSO 4 -induced cytochrome c release ( d ) and cleavage of caspase-3 and -9 ( e ). CASP: caspase. f Knockdown of FTL or FTH1 enhanced the CCCP/FeSO 4 -induced cleavage of caspase-3 and -9. g , h Knockdown of either caspase-3 or -9 abolished the CCCP/FeSO 4 -induced GSDME cleavage ( g ), pyroptosis and LDH release ( h ). Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques: Activation Assay, Confocal Microscopy

    ROS induces Tom20 oxidation in response to iron stimulation. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S were separately transfected into cells. a FeSO 4 induced Tom20 oxidation, which was attenuated by NAC even in the presence of CCCP. Western blotting was performed under reducing and non-reducing conditions to determine the Tom20 oxidation status. b Mutation of either Cys13 or Cys21 in Tom20 abolished the CCCP/FeSO 4 -induced Tom20 oxidation. c CCCP/FeSO 4 could not induce Tom20 C13S and Tom20 C21S accumulation. d Mutation of either Cys13 or Cys21 in Tom20 blocked mitochondrial aggregation as shown by confocal microscopy. Hsp60 represents the mitochondria, and DAPI was used to display the nuclei. e-h Mutation of either Cys13 or Cys21 in Tom20 abrogated the CCCP/FeSO 4 -induced cytochrome c release as detected in the cytosol fraction ( e ), cleavage of caspase-3 and -9 ( f ), LDH release and cell death ( g ), and reversed the cell morphology from pyroptosis to normal state ( h ). Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: ROS induces Tom20 oxidation in response to iron stimulation. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S were separately transfected into cells. a FeSO 4 induced Tom20 oxidation, which was attenuated by NAC even in the presence of CCCP. Western blotting was performed under reducing and non-reducing conditions to determine the Tom20 oxidation status. b Mutation of either Cys13 or Cys21 in Tom20 abolished the CCCP/FeSO 4 -induced Tom20 oxidation. c CCCP/FeSO 4 could not induce Tom20 C13S and Tom20 C21S accumulation. d Mutation of either Cys13 or Cys21 in Tom20 blocked mitochondrial aggregation as shown by confocal microscopy. Hsp60 represents the mitochondria, and DAPI was used to display the nuclei. e-h Mutation of either Cys13 or Cys21 in Tom20 abrogated the CCCP/FeSO 4 -induced cytochrome c release as detected in the cytosol fraction ( e ), cleavage of caspase-3 and -9 ( f ), LDH release and cell death ( g ), and reversed the cell morphology from pyroptosis to normal state ( h ). Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques: Transfection, Western Blot, Mutagenesis, Confocal Microscopy

    Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO 4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO 4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO 4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO 4 stimulation. c – e Knockdown of Tom20 reversed the CCCP/FeSO 4 -induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage ( c ), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 ( d ), and blocked mitochondria aggregation ( e ). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO 4 -induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO 4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO 4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO 4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO 4 stimulation. c – e Knockdown of Tom20 reversed the CCCP/FeSO 4 -induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage ( c ), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 ( d ), and blocked mitochondria aggregation ( e ). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO 4 -induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: The anti-tubulin (Cat# T-4026), anti-p62/SQSTM1 (Cat# WH0008878M1), anti-LC3 (Cat# L-7543), anti-GSDMD (Cat# G7422) and anti-Flag (Cat# F-1804) antibodies were purchased from Sigma.

    Techniques: Expressing

    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). β-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).

    Journal: PLoS ONE

    Article Title: The effect of “NutramilTM Complex,” food for special medical purpose, on breast and prostate carcinoma cells

    doi: 10.1371/journal.pone.0192860

    Figure Lengend Snippet: Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). β-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).

    Article Snippet: Subsequently, the immobilized proteins were incubated with the appropriate primary antibody: cytochrome c (#11940), Smac/Diablo (#2954), HtrA2/Omi (#9745) and β-Tubulin (#2128) (Cell Signaling Technology, MA, USA).

    Techniques: Expressing, Concentration Assay, Positive Control, Lysis, Protease Inhibitor, Incubation, Western Blot