f-actin Search Results


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  • 99
    Thermo Fisher β actin
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    Millipore monoclonal anti beta actin antibody
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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
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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
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    Abcam 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
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    Millipore anti 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
    Anti Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology beta actin
    Molecular analyses in the offspring liver (panel 2). (A) G6Pase and (B) PEPCK mRNA levels of the male and female offspring at 12-weeks old. Endogenous control <t>beta-actin</t> was used to normalize the expression of the selected genes. Data are expressed as the mean and SD (n = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P
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    Cytoskeleton Inc actin cytoskeleton
    Alterations in actin <t>cytoskeleton</t> status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p
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    Millipore anti β actin
    Alterations in actin <t>cytoskeleton</t> status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p
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    Millipore anti beta actin antibody mouse monoclonal
    Alterations in actin <t>cytoskeleton</t> status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p
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    Millipore mouse anti β actin
    Alterations in actin <t>cytoskeleton</t> status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p
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    Abcam anti β actin
    Alterations in actin <t>cytoskeleton</t> status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p
    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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    Millipore anti β actin antibody
    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and <t>β-actin</t> (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P
    Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam f actin
    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and <t>β-actin</t> (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P
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    Cell Signaling Technology Inc beta actin
    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and <t>β-actin</t> (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P
    Beta Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti actin
    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and <t>β-actin</t> (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P
    Mouse Anti Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 3797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti actin alpha smooth muscle
    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and <t>β-actin</t> (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P
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    Millipore alpha actin
    Morphology of 3D cell expansion on collagen scaffolds after two to three weeks in vitro culture in plastic wells; minced detrusor ((a)–(e)), detrusor and urothelium ((f)–(j)), and urothelium ((k)–(o)), compared to native pig bladder ((p)–(s)). Photomicrographs demonstrating routine staining with haematoxylin-eosin and immunostaining with <t>alpha-actin</t> after two and three weeks, respectively, for cytokeratin (MNF116) and Ki-67. Arrows (j, o, and s) indicating cells with a sustained proliferative capacity (Ki-67).
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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

    Repetitive magnetic stimulation treatment increased brain-derived neurotrophic factor (BDNF) expression in differentiated Neuro-2a cells. (A) The relative expression of BDNF was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR during neuronal differentiation of Neuro-2a cells. (B) Western blot analysis was performed using BDNF, and actin (as a control) antibodies in the Neuro-2a cells. (C) Comparison of relative protein expression for BDNF and actin (a control) in differentiated Neuro-2a cells with Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. *** p

    Journal: Frontiers in Neurology

    Article Title: High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway

    doi: 10.3389/fneur.2018.00285

    Figure Lengend Snippet: Repetitive magnetic stimulation treatment increased brain-derived neurotrophic factor (BDNF) expression in differentiated Neuro-2a cells. (A) The relative expression of BDNF was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR during neuronal differentiation of Neuro-2a cells. (B) Western blot analysis was performed using BDNF, and actin (as a control) antibodies in the Neuro-2a cells. (C) Comparison of relative protein expression for BDNF and actin (a control) in differentiated Neuro-2a cells with Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. *** p

    Article Snippet: Membranes were blocked and then incubated overnight at 4°C with anti-CaMKII (1:1,000 dilution, Abcam), anti-p-CREB (1:1,000 dilution, Santa Cruz Biotechnology), anti-BDNF (1:1,000 dilution, Abcam), and anti-ACTIN (1:5,000 dilution, Santacruz) antibodies.

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot, Software

    Validation of mRNA expression and protein quantification using qRT-PCR and western blot analysis in undifferentiated Neuro-2a cells. (A) The relative mRNA expression of target genes was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR. All results are expressed as means ± SEM. (B) Western blot analysis was performed using antibodies against calmodulin-dependent protein kinase II (CaMKII), phospho-cAMP response element binding (p-CREB), and actin (a control). All results are expressed as means ± SEM. (C) Comparison of relative protein expression for CaMKII, p-CREB, and actin (a control) with Multi Guage (v3.0) software (Fujifilm). * p

    Journal: Frontiers in Neurology

    Article Title: High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway

    doi: 10.3389/fneur.2018.00285

    Figure Lengend Snippet: Validation of mRNA expression and protein quantification using qRT-PCR and western blot analysis in undifferentiated Neuro-2a cells. (A) The relative mRNA expression of target genes was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR. All results are expressed as means ± SEM. (B) Western blot analysis was performed using antibodies against calmodulin-dependent protein kinase II (CaMKII), phospho-cAMP response element binding (p-CREB), and actin (a control). All results are expressed as means ± SEM. (C) Comparison of relative protein expression for CaMKII, p-CREB, and actin (a control) with Multi Guage (v3.0) software (Fujifilm). * p

    Article Snippet: Membranes were blocked and then incubated overnight at 4°C with anti-CaMKII (1:1,000 dilution, Abcam), anti-p-CREB (1:1,000 dilution, Santa Cruz Biotechnology), anti-BDNF (1:1,000 dilution, Abcam), and anti-ACTIN (1:5,000 dilution, Santacruz) antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Software

    Validation of mRNA expression and protein quantification using qRT-PCR and western blot analysis in differentiated Neuro-2a cells. (A) The relative expression of target genes was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR during neuronal differentiation of Neuro-2a cells. (B) Western blot analysis was performed with calmodulin-dependent protein kinase II (CaMKII), phospho-cAMP response element binding (p-CREB), and actin (as a control) antibodies in the Neuro-2a cells. (C) Comparison of relative protein expression for CaMKII, p-CREB, and actin (a control) in differentiated Neuro-2a cells using Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. * p

    Journal: Frontiers in Neurology

    Article Title: High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway

    doi: 10.3389/fneur.2018.00285

    Figure Lengend Snippet: Validation of mRNA expression and protein quantification using qRT-PCR and western blot analysis in differentiated Neuro-2a cells. (A) The relative expression of target genes was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR during neuronal differentiation of Neuro-2a cells. (B) Western blot analysis was performed with calmodulin-dependent protein kinase II (CaMKII), phospho-cAMP response element binding (p-CREB), and actin (as a control) antibodies in the Neuro-2a cells. (C) Comparison of relative protein expression for CaMKII, p-CREB, and actin (a control) in differentiated Neuro-2a cells using Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. * p

    Article Snippet: Membranes were blocked and then incubated overnight at 4°C with anti-CaMKII (1:1,000 dilution, Abcam), anti-p-CREB (1:1,000 dilution, Santa Cruz Biotechnology), anti-BDNF (1:1,000 dilution, Abcam), and anti-ACTIN (1:5,000 dilution, Santacruz) antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Software

    Repetitive magnetic stimulation treatment increased brain-derived neurotrophic factor (BDNF) expression in undifferentiated Neuro-2a cells. (A) The relative mRNA expression of BDNF was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR. All results are expressed as means ± SEM. (B) Western blot analysis was performed using antibodies against BDNF, and actin (a control). (C) Comparison of relative protein expression for BDNF and actin (a control) with Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. *** p

    Journal: Frontiers in Neurology

    Article Title: High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway

    doi: 10.3389/fneur.2018.00285

    Figure Lengend Snippet: Repetitive magnetic stimulation treatment increased brain-derived neurotrophic factor (BDNF) expression in undifferentiated Neuro-2a cells. (A) The relative mRNA expression of BDNF was normalized by sham expression and was calculated using the 2 −ΔΔCt method by qRT-PCR. All results are expressed as means ± SEM. (B) Western blot analysis was performed using antibodies against BDNF, and actin (a control). (C) Comparison of relative protein expression for BDNF and actin (a control) with Multi Guage (v3.0) software (Fujifilm). All results are expressed as means ± SEM. *** p

    Article Snippet: Membranes were blocked and then incubated overnight at 4°C with anti-CaMKII (1:1,000 dilution, Abcam), anti-p-CREB (1:1,000 dilution, Santa Cruz Biotechnology), anti-BDNF (1:1,000 dilution, Abcam), and anti-ACTIN (1:5,000 dilution, Santacruz) antibodies.

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot, Software

    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

    Molecular analyses in the offspring liver (panel 2). (A) G6Pase and (B) PEPCK mRNA levels of the male and female offspring at 12-weeks old. Endogenous control beta-actin was used to normalize the expression of the selected genes. Data are expressed as the mean and SD (n = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Journal: PLoS ONE

    Article Title: Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

    doi: 10.1371/journal.pone.0124737

    Figure Lengend Snippet: Molecular analyses in the offspring liver (panel 2). (A) G6Pase and (B) PEPCK mRNA levels of the male and female offspring at 12-weeks old. Endogenous control beta-actin was used to normalize the expression of the selected genes. Data are expressed as the mean and SD (n = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Article Snippet: Antibodies against sterol regulatory element binding protein-1c (SREBP-1c, 68 kDa), peroxisome proliferator-activated receptor alpha (PPAR-alpha, 55 kDa), fatty acid synthase (FAS, 270 kDa) and beta-actin (beta-actin, 43 kDa) were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Mouse Assay

    Molecular analyses in the offspring liver (panel 1). (A) SREBP-1c, (B) FAS, (C) PPAR-alpha and (D) CPT-1 mRNA levels of the male and female offspring at 12-weeks old. Endogenous control beta-actin was used to normalize the expression of the selected genes. Data are expressed as the mean and SD (N = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Journal: PLoS ONE

    Article Title: Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

    doi: 10.1371/journal.pone.0124737

    Figure Lengend Snippet: Molecular analyses in the offspring liver (panel 1). (A) SREBP-1c, (B) FAS, (C) PPAR-alpha and (D) CPT-1 mRNA levels of the male and female offspring at 12-weeks old. Endogenous control beta-actin was used to normalize the expression of the selected genes. Data are expressed as the mean and SD (N = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Article Snippet: Antibodies against sterol regulatory element binding protein-1c (SREBP-1c, 68 kDa), peroxisome proliferator-activated receptor alpha (PPAR-alpha, 55 kDa), fatty acid synthase (FAS, 270 kDa) and beta-actin (beta-actin, 43 kDa) were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA).

    Techniques: Cycling Probe Technology, Expressing, Mouse Assay

    Molecular analyses in the offspring liver (panel 3). Liver immunoblotting corrected by beta-actin expression of the male and female offspring at 12-weeks old. (A) SREBP-1c, (B) FAS, (C) PPAR-alpha, and (D) representative immunoblotting with bands (expressed in arbitrary units, a. u.). Data are expressed as the mean and SD (N = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Journal: PLoS ONE

    Article Title: Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

    doi: 10.1371/journal.pone.0124737

    Figure Lengend Snippet: Molecular analyses in the offspring liver (panel 3). Liver immunoblotting corrected by beta-actin expression of the male and female offspring at 12-weeks old. (A) SREBP-1c, (B) FAS, (C) PPAR-alpha, and (D) representative immunoblotting with bands (expressed in arbitrary units, a. u.). Data are expressed as the mean and SD (N = 5 mice per group, one-way ANOVA and the posthoc test of Holm–Sidak). Same letters represent equal groups with no statistical difference while different letters represent different groups from each other, with statistical difference ( P

    Article Snippet: Antibodies against sterol regulatory element binding protein-1c (SREBP-1c, 68 kDa), peroxisome proliferator-activated receptor alpha (PPAR-alpha, 55 kDa), fatty acid synthase (FAS, 270 kDa) and beta-actin (beta-actin, 43 kDa) were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Mouse Assay

    Scheme of the mechanisms obtained in the study. Liver steatosis observed in offspring of obese father and obese mother is possibly the result of different activated pathways, resulting from a framework of hyperinsulinemia. It is suggested that the paternal obesity intensifies the lipogenesis pathway through increased gene and protein expression of SREBP-1c, which stimulates the synthesis of fatty acids by the FAS enzyme. Maternal obesity led to a less intense lipogenesis and decreased beta-oxidation showed by a lower gene and protein expression of PPAR alpha and CPT-1. Arrow-up (↑) indicates an increase and arrow-down (↓) indicates a decrease in both gene and protein expressions. Abbreviations: sterol regulating element binding protein (SREBP-1c); fatty acid synthase (FAS); peroxisome proliferator activator receptor alpha (PPAR-alpha); carnitine palmitoyltransferase I (CPT-1); free fatty acid (FFA).

    Journal: PLoS ONE

    Article Title: Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

    doi: 10.1371/journal.pone.0124737

    Figure Lengend Snippet: Scheme of the mechanisms obtained in the study. Liver steatosis observed in offspring of obese father and obese mother is possibly the result of different activated pathways, resulting from a framework of hyperinsulinemia. It is suggested that the paternal obesity intensifies the lipogenesis pathway through increased gene and protein expression of SREBP-1c, which stimulates the synthesis of fatty acids by the FAS enzyme. Maternal obesity led to a less intense lipogenesis and decreased beta-oxidation showed by a lower gene and protein expression of PPAR alpha and CPT-1. Arrow-up (↑) indicates an increase and arrow-down (↓) indicates a decrease in both gene and protein expressions. Abbreviations: sterol regulating element binding protein (SREBP-1c); fatty acid synthase (FAS); peroxisome proliferator activator receptor alpha (PPAR-alpha); carnitine palmitoyltransferase I (CPT-1); free fatty acid (FFA).

    Article Snippet: Antibodies against sterol regulatory element binding protein-1c (SREBP-1c, 68 kDa), peroxisome proliferator-activated receptor alpha (PPAR-alpha, 55 kDa), fatty acid synthase (FAS, 270 kDa) and beta-actin (beta-actin, 43 kDa) were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Cycling Probe Technology, Binding Assay

    Alterations in actin cytoskeleton status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

    doi: 10.1074/jbc.M111.253153

    Figure Lengend Snippet: Alterations in actin cytoskeleton status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p

    Article Snippet: Stabilization of the actin cytoskeleton with jasplakinolide (30 nm , 1-h treatment at room temperature) had no effect on P2X1 receptor current rundown in whole cell recordings , suggesting that rundown does not result from a disruption of the actin cytoskeleton.

    Techniques: Expressing

    Differential sensitivity of P2X receptors to actin cytoskeleton disruption. The effect of actin cytoskeleton disruption by cytochalasin D was tested on currents mediated by P2X2, P2X3, P2X4, P2X5, and P2X7 receptors transiently transfected in HEK293 cells. Currents were evoked by application of ATP (100 μ m ) or BzATP (100 μ m in 0.3 m m CaCl 2 , 0 m m MgCl 2 containing solution) for the P2X7 receptor. A , representative traces of ATP-evoked currents of P2X receptors from control cells ( left trace ) and cells treated with cytochalasin D ( right trace ) (500 n m , 1 h). B , summary of the effects of actin cytoskeleton disruption for currents mediated by P2X2, P2X3, P2X4, P2X5, and P2X7 receptors ( n = 8–14). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

    doi: 10.1074/jbc.M111.253153

    Figure Lengend Snippet: Differential sensitivity of P2X receptors to actin cytoskeleton disruption. The effect of actin cytoskeleton disruption by cytochalasin D was tested on currents mediated by P2X2, P2X3, P2X4, P2X5, and P2X7 receptors transiently transfected in HEK293 cells. Currents were evoked by application of ATP (100 μ m ) or BzATP (100 μ m in 0.3 m m CaCl 2 , 0 m m MgCl 2 containing solution) for the P2X7 receptor. A , representative traces of ATP-evoked currents of P2X receptors from control cells ( left trace ) and cells treated with cytochalasin D ( right trace ) (500 n m , 1 h). B , summary of the effects of actin cytoskeleton disruption for currents mediated by P2X2, P2X3, P2X4, P2X5, and P2X7 receptors ( n = 8–14). ***, p

    Article Snippet: Stabilization of the actin cytoskeleton with jasplakinolide (30 nm , 1-h treatment at room temperature) had no effect on P2X1 receptor current rundown in whole cell recordings , suggesting that rundown does not result from a disruption of the actin cytoskeleton.

    Techniques: Transfection

    Inhibition of P2X1 receptor-mediated currents by actin cytoskeleton disruption. Effects of actin cytoskeleton disruption by cytochalasin D ( Cyto ) or latrunculin A ( Lat ) on P2X1 receptor currents were tested for recombinant human P2X1 receptors expressed in HEK293 cells ( A , C , E , and F ) and native rat smooth muscle P2X1 receptors ( D ). Application of cytochalasin D (5 μ m , gray symbols ) in external solution reduced the amplitude of α,β-meATP-evoked currents (10 μ m , applied every 5 min) in the permeabilized patch recording configuration ( A and B ). 20-min treatment with cytochalasin D before stimulating the P2X1 receptor is shown as a star . In the whole cell recording configuration P2X1 receptor currents in HEK293 cells or rat smooth muscle cells treated with cytochalasin D (500 n m ,1 h) or latrunculin A (500 n m , 1 h) were reduced ( C–E ). Surface biotinylation showed that actin cytoskeleton disruption by cytochalasin D (500 n m ,1 h) did not change either the total or surface level of P2X1 receptors expression in HEK293 cells ( F ). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

    doi: 10.1074/jbc.M111.253153

    Figure Lengend Snippet: Inhibition of P2X1 receptor-mediated currents by actin cytoskeleton disruption. Effects of actin cytoskeleton disruption by cytochalasin D ( Cyto ) or latrunculin A ( Lat ) on P2X1 receptor currents were tested for recombinant human P2X1 receptors expressed in HEK293 cells ( A , C , E , and F ) and native rat smooth muscle P2X1 receptors ( D ). Application of cytochalasin D (5 μ m , gray symbols ) in external solution reduced the amplitude of α,β-meATP-evoked currents (10 μ m , applied every 5 min) in the permeabilized patch recording configuration ( A and B ). 20-min treatment with cytochalasin D before stimulating the P2X1 receptor is shown as a star . In the whole cell recording configuration P2X1 receptor currents in HEK293 cells or rat smooth muscle cells treated with cytochalasin D (500 n m ,1 h) or latrunculin A (500 n m , 1 h) were reduced ( C–E ). Surface biotinylation showed that actin cytoskeleton disruption by cytochalasin D (500 n m ,1 h) did not change either the total or surface level of P2X1 receptors expression in HEK293 cells ( F ). ***, p

    Article Snippet: Stabilization of the actin cytoskeleton with jasplakinolide (30 nm , 1-h treatment at room temperature) had no effect on P2X1 receptor current rundown in whole cell recordings , suggesting that rundown does not result from a disruption of the actin cytoskeleton.

    Techniques: Inhibition, Recombinant, Expressing

    Stabilization of actin polymerization prevents disruption of lipid rafts that are essential for P2X1 receptors function. A , representative traces of P2X1 receptor-mediated currents evoked by application of α,β-meATP (10 m m ) for nontreated cells and after incubation with jasplakinolide ( Jasp , 30 n m , 1 h), Mβ-CD (10 m m , 1 h), or jasplakinolide (30 n m ) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h). B , summary data of actin polymerization and disruption of lipid rafts on P2X1 receptor-mediated currents. P2X1 receptor currents were unaffected by treatment with jasplakinolide (30 n m , 1 h) which stabilizes polymerized actin cytoskeleton ( n = 9). Mβ-CD (10 m m , 1 h) treatment reduced the peak amplitude of P2X1 receptor currents by > 90% ( n = 15). Treatment with jasplakinolide (30 n m , 1 h) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h) abolished the effect of Mβ-CD ( n = 13). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

    doi: 10.1074/jbc.M111.253153

    Figure Lengend Snippet: Stabilization of actin polymerization prevents disruption of lipid rafts that are essential for P2X1 receptors function. A , representative traces of P2X1 receptor-mediated currents evoked by application of α,β-meATP (10 m m ) for nontreated cells and after incubation with jasplakinolide ( Jasp , 30 n m , 1 h), Mβ-CD (10 m m , 1 h), or jasplakinolide (30 n m ) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h). B , summary data of actin polymerization and disruption of lipid rafts on P2X1 receptor-mediated currents. P2X1 receptor currents were unaffected by treatment with jasplakinolide (30 n m , 1 h) which stabilizes polymerized actin cytoskeleton ( n = 9). Mβ-CD (10 m m , 1 h) treatment reduced the peak amplitude of P2X1 receptor currents by > 90% ( n = 15). Treatment with jasplakinolide (30 n m , 1 h) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h) abolished the effect of Mβ-CD ( n = 13). ***, p

    Article Snippet: Stabilization of the actin cytoskeleton with jasplakinolide (30 nm , 1-h treatment at room temperature) had no effect on P2X1 receptor current rundown in whole cell recordings , suggesting that rundown does not result from a disruption of the actin cytoskeleton.

    Techniques: Incubation

    Stabilization of the actin cytoskeleton by jasplakinolide does not prevent the rundown of P2X1 receptor-mediated currents in the whole cell recording configuration. In the permeabilized patch recording configuration, which supports intracellular signaling pathways, reproducible α,β-meATP (10 μ m )-evoked currents were observed every 5 min both for control cells and cells treated with jasplakinolide (30 n m , 1 h) to stabilize the actin cytoskeleton. In the whole cell recording configuration P2X1 receptor currents decreased in amplitude following repeated application of agonist at 5-min intervals. This rundown was unaffected by jasplakinolide treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

    doi: 10.1074/jbc.M111.253153

    Figure Lengend Snippet: Stabilization of the actin cytoskeleton by jasplakinolide does not prevent the rundown of P2X1 receptor-mediated currents in the whole cell recording configuration. In the permeabilized patch recording configuration, which supports intracellular signaling pathways, reproducible α,β-meATP (10 μ m )-evoked currents were observed every 5 min both for control cells and cells treated with jasplakinolide (30 n m , 1 h) to stabilize the actin cytoskeleton. In the whole cell recording configuration P2X1 receptor currents decreased in amplitude following repeated application of agonist at 5-min intervals. This rundown was unaffected by jasplakinolide treatment.

    Article Snippet: Stabilization of the actin cytoskeleton with jasplakinolide (30 nm , 1-h treatment at room temperature) had no effect on P2X1 receptor current rundown in whole cell recordings , suggesting that rundown does not result from a disruption of the actin cytoskeleton.

    Techniques:

    Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and β-actin (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P

    Journal: Oncoimmunology

    Article Title: Glioma-induced SIRT1-dependent activation of hMOF histone H4 lysine 16 acetyltransferase in microglia promotes a tumor supporting phenotype

    doi: 10.1080/2162402X.2017.1382790

    Figure Lengend Snippet: Glioma‐induced microglia tumor‐supporting phenotype is associated with increased H4K16 acetylation. (A) qPCR analysis of Il1β, Il6, Mmp14, Ccl22, Chil3 and Nos2 mRNA expression in BV2 microglia grown as monocultures or with C6 glioma cells as segregated cocultures (key); results are presented relative to those of each monoculture, set as 1. (B) Immunoblot analysis of MMP14 and β-actin (right margin, molecular size, in kilodaltons (kDa)) with quantification (C) in BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia monoculture, set as 1. (D) Quantification of the migration of C6 glioma cells in Transwells with BV2 microglia or C6-cocultured BV2 microglia in the lower compartment; results are presented relative to those of C6 cells alone, set as 1. (E and F) Immunoblot analysis of H4K16ac and β-actin (right margin, molecular size, in kilodaltons (kDa)) (E) with quantification (F) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented to relative to those of BV2 microglia control (0 h), set as 1. (G) Confocal microscopy of BV2 microglia cultured for 4 h as a monoculture or with C6 glioma cells as segregated coculture, with immunostaining for H4K16ac and Hoechst nuclear counterstain. Scale bar, 10 μm. ns, non-significant, *P

    Article Snippet: Immunoblot with anti-β-actin antibody (1:3000; A-3853, Sigma Aldrich) was used for standardization of protein loading.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Migration, Confocal Microscopy, Immunostaining

    Nuclear relocalisation of microglial SIRT1 is observed upon glioma coculture. (A to D) Immunoblot analysis of SIRT1 (A) or hMOF (C) and β-actin (A and C) with quantification (B and D) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented relative to those of BV2 microglia control (0 h), set as 1. (E) Immunoblot analysis of SIRT1, actin and lamin β1 lysate in subcellular fractions of BV2 microglia grown for 4 h as monocultures or with C6 cells as segregated cocultures. (F) Quantification of SIRT1 in subcellular fractions of e; results are presented relative to those of BV2 monocultures, set as 1. (G) Confocal microscopy of BV2 microglia grown for 4 h as monocultures or with C6 cells as segregated cocultures, with immunostaining for SIRT1 and Hoechst nuclear counterstain. Last row of the depicted pictures are higher magnification of the merged confocal images. Scale bar, 20 µm. *P

    Journal: Oncoimmunology

    Article Title: Glioma-induced SIRT1-dependent activation of hMOF histone H4 lysine 16 acetyltransferase in microglia promotes a tumor supporting phenotype

    doi: 10.1080/2162402X.2017.1382790

    Figure Lengend Snippet: Nuclear relocalisation of microglial SIRT1 is observed upon glioma coculture. (A to D) Immunoblot analysis of SIRT1 (A) or hMOF (C) and β-actin (A and C) with quantification (B and D) in BV2 microglia control (0 h) or cultured for 2 h, 4 h or 6 h as monoculture or with C6 glioma cells as segregated coculture; results are presented relative to those of BV2 microglia control (0 h), set as 1. (E) Immunoblot analysis of SIRT1, actin and lamin β1 lysate in subcellular fractions of BV2 microglia grown for 4 h as monocultures or with C6 cells as segregated cocultures. (F) Quantification of SIRT1 in subcellular fractions of e; results are presented relative to those of BV2 monocultures, set as 1. (G) Confocal microscopy of BV2 microglia grown for 4 h as monocultures or with C6 cells as segregated cocultures, with immunostaining for SIRT1 and Hoechst nuclear counterstain. Last row of the depicted pictures are higher magnification of the merged confocal images. Scale bar, 20 µm. *P

    Article Snippet: Immunoblot with anti-β-actin antibody (1:3000; A-3853, Sigma Aldrich) was used for standardization of protein loading.

    Techniques: Cell Culture, Confocal Microscopy, Immunostaining

    Morphology of 3D cell expansion on collagen scaffolds after two to three weeks in vitro culture in plastic wells; minced detrusor ((a)–(e)), detrusor and urothelium ((f)–(j)), and urothelium ((k)–(o)), compared to native pig bladder ((p)–(s)). Photomicrographs demonstrating routine staining with haematoxylin-eosin and immunostaining with alpha-actin after two and three weeks, respectively, for cytokeratin (MNF116) and Ki-67. Arrows (j, o, and s) indicating cells with a sustained proliferative capacity (Ki-67).

    Journal: BioMed Research International

    Article Title: Expansion of Submucosal Bladder Wall Tissue In Vitro and In Vivo

    doi: 10.1155/2016/5415012

    Figure Lengend Snippet: Morphology of 3D cell expansion on collagen scaffolds after two to three weeks in vitro culture in plastic wells; minced detrusor ((a)–(e)), detrusor and urothelium ((f)–(j)), and urothelium ((k)–(o)), compared to native pig bladder ((p)–(s)). Photomicrographs demonstrating routine staining with haematoxylin-eosin and immunostaining with alpha-actin after two and three weeks, respectively, for cytokeratin (MNF116) and Ki-67. Arrows (j, o, and s) indicating cells with a sustained proliferative capacity (Ki-67).

    Article Snippet: The tissue specimens were further evaluated by immunostaining with primary antibodies against various cytokeratins (1 : 300) (pancytokeratins 1–7, 10, 13–16, and 19, DakoCytomation, Cat#M0821, clone: MNF 116), uroplakin III (1 : 400) (Fitzgerald Industries, Clone AU1, CatRDI-PRO610108), alpha-actin (1 : 10 000) (α Smooth Muscle, Sigma-Aldrich, clone 1A4, purified mouse immunoglobulin, product number A 5228), and Ki-67 (1 : 200) (Ki-67; Lab Vision, Rabbit Monoclonal, clone SP6).

    Techniques: In Vitro, Staining, Immunostaining

    Morphology after four weeks in vivo . Transplants with minced detrusor ((a)–(d)), detrusor and urothelium ((e)–(h)), urothelium ((i)–(l)), and control ((m)–(p)). Photomicrographs demonstrating routine histology with haematoxylin-eosin and Masson's trichrome and immunostaining for cytokeratin (MNF116) ((c), (g), and (o)), uroplakin III (k), or alpha-actin, with L indicating the lumen. Regenerated detrusor muscle indicated by ( ∗ ), separated from the shivering muscle marked with (†) ((b) and (f)). (j) No regenerated detrusor muscle was shown when transplanting with only minced urothelium. (k) A multilayered continuous transitional epithelium of urothelial origin was confirmed by uroplakin in specimens with urothelium. Immunostaining with alpha-actin demonstrated smooth muscle cells in specimens transplanted with minced detrusor muscle (d) as well as minced detrusor and urothelium (h).

    Journal: BioMed Research International

    Article Title: Expansion of Submucosal Bladder Wall Tissue In Vitro and In Vivo

    doi: 10.1155/2016/5415012

    Figure Lengend Snippet: Morphology after four weeks in vivo . Transplants with minced detrusor ((a)–(d)), detrusor and urothelium ((e)–(h)), urothelium ((i)–(l)), and control ((m)–(p)). Photomicrographs demonstrating routine histology with haematoxylin-eosin and Masson's trichrome and immunostaining for cytokeratin (MNF116) ((c), (g), and (o)), uroplakin III (k), or alpha-actin, with L indicating the lumen. Regenerated detrusor muscle indicated by ( ∗ ), separated from the shivering muscle marked with (†) ((b) and (f)). (j) No regenerated detrusor muscle was shown when transplanting with only minced urothelium. (k) A multilayered continuous transitional epithelium of urothelial origin was confirmed by uroplakin in specimens with urothelium. Immunostaining with alpha-actin demonstrated smooth muscle cells in specimens transplanted with minced detrusor muscle (d) as well as minced detrusor and urothelium (h).

    Article Snippet: The tissue specimens were further evaluated by immunostaining with primary antibodies against various cytokeratins (1 : 300) (pancytokeratins 1–7, 10, 13–16, and 19, DakoCytomation, Cat#M0821, clone: MNF 116), uroplakin III (1 : 400) (Fitzgerald Industries, Clone AU1, CatRDI-PRO610108), alpha-actin (1 : 10 000) (α Smooth Muscle, Sigma-Aldrich, clone 1A4, purified mouse immunoglobulin, product number A 5228), and Ki-67 (1 : 200) (Ki-67; Lab Vision, Rabbit Monoclonal, clone SP6).

    Techniques: In Vivo, Immunostaining