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moi-multiplicity-infection

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A visual protocol for pseudoislet formation with viral transduction for gen...
Published: 19 May 2021
Figure 1 A visual protocol for pseudoislet formation with viral transduction for genetic modification studies. hICM, human islet culture media; MOI, multiplicity of infection; RT-qPCR, quantitative RT-PCR. Figure 1. A visual protocol for pseudoislet formation with viral transduction for genetic ... More
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mRNA induction of antiviral genes, cytokines, and chemokines in human pancr...
Published: 17 July 2012
FIG. 1. mRNA induction of antiviral genes, cytokines, and chemokines in human pancreatic islets upon CVB3 infection. A: Human islets were infected at a multiplicity of infection (MOI) of 10, and virus titers were determined by end point titration at the indicated times pi. B: Images of islets taken 48 h pi. C: Human islets were infected with CVB3 or UV-inactivated CVB3 (MOI 10) or were mock infected, and at indicated times, mRNA induction of indicated genes was determined using qPCR. D: mRNA induction of indicated genes was determined by qPCR in islets infected as in C. Data are representative examples of at least three experiments using different donors. AU, arbitrary unit. FIG. 1. mRNA induction of antiviral genes, cytokines, and chemokines in human pancreatic islets upon CVB3 infection. A: Human islets were infected at a multiplicity of infection (MOI) of 10, and virus titers were determined by end point titration at the indicated times pi. B: Images of islets taken 48 h pi. C: Human islets were infected with CVB3 or UV-inactivated CVB3 (MOI 10) or were mock infected, and at indicated times, mRNA induction of indicated genes was determined using qPCR. D: mRNA induction of indicated genes was determined by qPCR in islets infected as in C. Data are representative examples of at least three experiments using different donors. AU, arbitrary unit. More
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Comparison of islet cell transduction efficiency with low <span class="search-highlight">multiplicities</span> of...
Published: 01 March 2001
FIG. 4. Comparison of islet cell transduction efficiency with low multiplicities of either rAAV2 or rAAV5. Murine islets were plated at ∼50 intact islets per well (1 × 105 cells per well), infected with Ad5 at an MOI of 10, and then infected with rAAV2-RSVnlacZ (A) or rAAV5-RSVnlacZ (B) at an MOI of 100. Cells were cultured for 48 h and then stained with X-gal. Transduced cells demonstrate blue-stained nuclei. FIG. 4. Comparison of islet cell transduction efficiency with low multiplicities of either rAAV2 or rAAV5. Murine islets were plated at ∼50 intact islets per well (1 × 105 cells per well), infected with Ad5 at an MOI of 10, and then infected with rAAV2-RSVnlacZ (A) or rAAV5-RSVnlacZ (B) at an MOI of 100. Cells were cultured for 48 h and then stained with X-gal. Transduced cells demonstrate blue-stained nuclei. More
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Adenovirus-mediated dose-dependent overexpression of h1-calponin protein in...
Published: 01 March 2003
FIG. 5. Adenovirus-mediated dose-dependent overexpression of h1-calponin protein in AR42J-B13 cells. A: Cells were infected with Ad-calponin for 3 h at multiplicity of infection (MOI) indicated and then incubated for 48 h. Total cell lysates were extracted and analyzed for h1-calponin by Western blot analysis. B: Western blot analysis for h1-calponin in total cell lysates from AR42J cells (lane 1), AR42J cells infected with Ad-calponin for 24 h (lane 2), human umbilical smooth muscle cells (lane 3), and A7r5 rat smooth muscle cells (lane 4). C: Cells were infected with Ad-LacZ (upper panel) or Ad-calponin (lower panel) at MOI of 30 and then incubated with (“+”) or without (“-”) activin A and HGF for the times indicated. Expression of h1-calponin protein was analyzed by Western blot analysis. FIG. 5. Adenovirus-mediated dose-dependent overexpression of h1-calponin protein in AR42J-B13 cells. A: Cells were infected with Ad-calponin for 3 h at multiplicity of infection (MOI) indicated and then incubated for 48 h. Total cell lysates were extracted and analyzed for h1-calponin by Western blot analysis. B: Western blot analysis for h1-calponin in total cell lysates from AR42J cells (lane 1), AR42J cells infected with Ad-calponin for 24 h (lane 2), human umbilical smooth muscle cells (lane 3), and A7r5 rat smooth muscle cells (lane 4). C: Cells were infected with Ad-LacZ (upper panel) or Ad-calponin (lower panel) at MOI of 30 and then incubated with (“+”) or without (“-”) activin A and HGF for the times indicated. Expression of h1-calponin protein was analyzed by Western blot analysis. More
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Regulation by KLF15 of genes related to gluconeogenesis or amino acid degra...
Published: 14 April 2010
FIG. 1. Regulation by KLF15 of genes related to gluconeogenesis or amino acid degradation. A: Rat primary cultured hepatocytes that had been infected with an adenovirus encoding KLF15 shRNA (shKLF15) or with a control virus containing the U6 gene promoter alone (Cont) at a multiplicity of infection (MOI) of 10 pfu per cell were incubated for 6 h in the presence of the cell-permeable cAMP analog 8-CPT-cAMP at 100 μmol/l. The abundance of the indicated mRNAs was then determined by RT and real-time PCR analysis. B: HL1c rat hepatocytes that had been infected with adenoviruses encoding KLF15, PGC1α, or LacZ at the indicated MOIs (plaque-forming units per cells) were subjected to immunoblot (IB) analysis of PGC1α and KLF15 (left panel) or to RT and real-time PCR analysis of PEPCK and HPD mRNAs (middle and right panels). C: HL1c cells that had been infected (or not) with adenoviruses either encoding KLF15 shRNA, containing the U6 promoter alone (Cont), or encoding PGC1α (each at an MOI of 10 pfu/cell) were subjected to RT and real-time PCR analysis of the indicated mRNAs. All quantitative data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01. FIG. 1. Regulation by KLF15 of genes related to gluconeogenesis or amino acid degradation. A: Rat primary cultured hepatocytes that had been infected with an adenovirus encoding KLF15 shRNA (shKLF15) or with a control virus containing the U6 gene promoter alone (Cont) at a multiplicity of infection (MOI) of 10 pfu per cell were incubated for 6 h in the presence of the cell-permeable cAMP analog 8-CPT-cAMP at 100 μmol/l. The abundance of the indicated mRNAs was then determined by RT and real-time PCR analysis. B: HL1c rat hepatocytes that had been infected with adenoviruses encoding KLF15, PGC1α, or LacZ at the indicated MOIs (plaque-forming units per cells) were subjected to immunoblot (IB) analysis of PGC1α and KLF15 (left panel) or to RT and real-time PCR analysis of PEPCK and HPD mRNAs (middle and right panels). C: HL1c cells that had been infected (or not) with adenoviruses either encoding KLF15 shRNA, containing the U6 promoter alone (Cont), or encoding PGC1α (each at an MOI of 10 pfu/cell) were subjected to RT and real-time PCR analysis of the indicated mRNAs. All quantitative data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01. More
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GSK3β regulation of nSREBP-1 and GIRK4 levels in chick atrial myocytes. ...
Published: 15 May 2014
Figure 5 GSK3β regulation of nSREBP-1 and GIRK4 levels in chick atrial myocytes. A: Western blots demonstrate dose dependence of Kenpaullone inhibition of GSK3β on GIRK4 and nSREBP-1 levels. B: Densitometric analysis of Western blots similar to those in A, normalized to β-actin. C: Western blot analysis of levels of GIRK4 and nSREBP-1 protein in atrial myocytes infected with increasing multiplicity of infection (MOI) of an adenovirus expressing an HA-tagged DA-GSK3β. D: Densitometric analysis of Western blots similar to those in C. E: Effect of adenoviral expression of Myr-Akt on the phosphorylation of GSK3β and levels of nSREBP-1 and GIRK4 proteins. Chick atrial myocytes were infected with Ad-Myr-Akt or Ad-GFP at an MOI of 50 pfu/cell for 3 h, followed by incubation for 48 h in fresh medium. Cells were harvested, and levels of nSREBP-1, GIRK4, and p-GSK3β were determined. F: Densitometric analysis of Western blots similar to those in E. Data are normalized to β-actin. *P < 0.05, **P < 0.01. Figure 5. GSK3β regulation of nSREBP-1 and GIRK4 levels in chick atrial myocytes. A: Western blots demonstrate dose dependence of Kenpaullone inhibition of GSK3β on GIRK4 and nSREBP-1 levels. B: Densitometric analysis of Western blots similar to those in A, normalized to β-actin. C: Western blot analysis of levels of GIRK4 and nSREBP-1 protein in atrial myocytes infected with increasing multiplicity of infection (MOI) of an adenovirus expressing an HA-tagged DA-GSK3β. D: Densitometric analysis of Western blots similar to those in C. E: Effect of adenoviral expression of Myr-Akt on the phosphorylation of GSK3β and levels of nSREBP-1 and GIRK4 proteins. Chick atrial myocytes were infected with Ad-Myr-Akt or Ad-GFP at an MOI of 50 pfu/cell for 3 h, followed by incubation for 48 h in fresh medium. Cells were harvested, and levels of nSREBP-1, GIRK4, and p-GSK3β were determined. F: Densitometric analysis of Western blots similar to those in E. Data are normalized to β-actin. *P < 0.05, **P < 0.01. More
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Activation of Rap1 is required for the augmenting effect of GLP-1 or 8-pCPT...
Published: 14 October 2014
Figure 5 Activation of Rap1 is required for the augmenting effect of GLP-1 or 8-pCPT and glibenclamide (GLB) on insulin secretion. A: Domain structures of full-length Epac2A and Epac2A(465–1011). A, cNBD-A; DEP, Dishevelled, Egl-10, and Pleckstrin domain; B, cNBD-B; REM, Ras exchange motif; RA, Ras association domain; CDC25-HD, CDC25 homology domain. B: Rap1 activation in noninfected MIN6-K8 cells or MIN6-K8 cells infected with adenovirus carrying mCherry or Epac2A(465–1011) at a multiplicity of infection (MOI) of 10, 30, or 100. C: Insulin secretion from noninfected MIN6-K8 cells or MIN6-K8 cells infected with adenovirus carrying mCherry or Epac2A(465–1011) at an MOI of 10, 30, or 100. Data are expressed as means ± SEM (n = 4 for each group). *P < 0.05, **P < 0.01 (Tukey-Kramer test). D: Effects of Rap1 knockdown on insulin secretion from MIN6-K8 cells. Top: Efficiency of knockdown was confirmed by immunoblot analysis. Bottom: Insulin secretion from MIN6-K8 cells transfected with nontarget siRNA or siRNA for Rap1a and Rap1b. Data are expressed as means ± SEM (n = 4 for each group). *P < 0.005, **P < 0.001 (Tukey-Kramer test). Figure 5. Activation of Rap1 is required for the augmenting effect of GLP-1 or 8-pCPT and glibenclamide (GLB) on insulin secretion. A: Domain structures of full-length Epac2A and Epac2A(465–1011). A, cNBD-A; DEP, Dishevelled, Egl-10, and Pleckstrin domain; B, cNBD-B; REM, Ras exchange motif; RA, Ras association domain; CDC25-HD, CDC25 homology domain. B: Rap1 activation in noninfected MIN6-K8 cells or MIN6-K8 cells infected with adenovirus carrying mCherry or Epac2A(465–1011) at a multiplicity of infection (MOI) of 10, 30, or 100. C: Insulin secretion from noninfected MIN6-K8 cells or MIN6-K8 cells infected with adenovirus carrying mCherry or Epac2A(465–1011) at an MOI of 10, 30, or 100. Data are expressed as means ± SEM (n = 4 for each group). *P < 0.05, **P < 0.01 (Tukey-Kramer test). D: Effects of Rap1 knockdown on insulin secretion from MIN6-K8 cells. Top: Efficiency of knockdown was confirmed by immunoblot analysis. Bottom: Insulin secretion from MIN6-K8 cells transfected with nontarget siRNA or siRNA for Rap1a and Rap1b. Data are expressed as means ± SEM (n = 4 for each group). *P < 0.005, **P < 0.001 (Tukey-Kramer test). More
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GDF11 alleviates EPC apoptosis in vitro. EPCs were pretreated with SB431542...
Published: 19 July 2018
Figure 5 GDF11 alleviates EPC apoptosis in vitro. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 24-h incubation with HG. A: After incubation, apoptosis of EPCs was stained with Annexin V-FITC and propidium iodide and assessed by flow cytometry. B: Quantitative analysis of A. C and D: Representative immunoblots and densitometric quantification for the expressions of the proteins Bcl-2, Bax, and cleaved-caspase3. Data are mean ± SEM for five independent experiments. *P < 0.05, **P < 0.01. MOI, multiplicity of infection. Figure 5. GDF11 alleviates EPC apoptosis in vitro. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 24-h incubation with HG. A: After incubation, apoptosis of EPCs was stained with Annexin V-FITC and propidium iodide and assessed by flow cytometry. B: Quantitative analysis of A. C and D: Representative immunoblots and densitometric quantification for the expressions of the proteins Bcl-2, Bax, and cleaved-caspase3. Data are mean ± SEM for five independent experiments. *P < 0.05, **P < 0.01. MOI, multiplicity of infection. More
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A negative regulation of Nrf2 by ERK worsens insulin sensitivity in HL-1 ce...
Published: 21 January 2011
FIG. 3. A negative regulation of Nrf2 by ERK worsens insulin sensitivity in HL-1 cells. A: Insulin activates Nrf2 via a negative cross-talk between PI3K and ERK. Cells were transfected with ARE-luc and pRL-TK-luc in Opti-MEM (Invitrogen) for 6 h and then changed with 0.1% FBS medium in the absence of norepinephrine for 48 h. The cells were next stimulated with 100 nmol/L of insulin, 10 μmol/L of U0126, and 10 μmol/L of LY294002 as indicated for 12 h. Nrf2 transcriptional activity was measured by a dual luciferase assay kit (Promega). *P < 0.05 vs. control (−), #P < 0.05 vs. insulin (+); n = 4. B: Effect of overexpression of Nrf2 on insulin sensitivity in HL-1 cells. Cells infected with Ad-βGal (20 multiplicity of infection [MOI]) and Ad-Nrf2 (20 MOI) were pretreated with H2O2 (100 μmol/L) as in Fig. 1 and subjected to 2-DG uptake assay. *P < 0.05 vs. control (−), n = 4. C: Infected cells were pretreated as in Fig. 1 and stimulated with or without insulin (100 nmol/L) for 10 min. *P < 0.05 vs. control (H2O2− and insulin−); otherwise statistical difference was indicated. FIG. 3. A negative regulation of Nrf2 by ERK worsens insulin sensitivity in HL-1 cells. A: Insulin activates Nrf2 via a negative cross-talk between PI3K and ERK. Cells were transfected with ARE-luc and pRL-TK-luc in Opti-MEM (Invitrogen) for 6 h and then changed with 0.1% FBS medium in the absence of norepinephrine for 48 h. The cells were next stimulated with 100 nmol/L of insulin, 10 μmol/L of U0126, and 10 μmol/L of LY294002 as indicated for 12 h. Nrf2 transcriptional activity was measured by a dual luciferase assay kit (Promega). *P < 0.05 vs. control (−), #P < 0.05 vs. insulin (+); n = 4. B: Effect of overexpression of Nrf2 on insulin sensitivity in HL-1 cells. Cells infected with Ad-βGal (20 multiplicity of infection [MOI]) and Ad-Nrf2 (20 MOI) were pretreated with H2O2 (100 μmol/L) as in Fig. 1 and subjected to 2-DG uptake assay. *P < 0.05 vs. control (−), n = 4. C: Infected cells were pretreated as in Fig. 1 and stimulated with or without insulin (100 nmol/L) for 10 min. *P < 0.05 vs. control (H2O2− and insulin−); otherwise statistical difference was indicated. More
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PPP1R3G stimulates glycogen synthesis in hepatocytes. <em>A</em>: P...
Published: 23 April 2011
FIG. 3. PPP1R3G stimulates glycogen synthesis in hepatocytes. A: Primary hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 multiplicity of infection (MOI) of Ad-GFP or Ad-PPP1R3G with a Flag tag at the N-terminus. Twenty-four hours later, the cells were harvested for immunoblotting with antibodies as indicated. B: Primary hepatocytes isolated from overnight fasted mice were infected with Ad-GFP or Ad-PPP1R3G at different MOI, followed by glycogen measurement. ***P < 0.001 in comparison with the Ad-GFP group. C: The cells as in A were treated for 24 h with various glucose concentrations, followed by measurement of glycogen content. Data are shown as mean ± SD. **P < 0.01 as comparison between the two groups. D and E: The cells as in A were treated with 100 nmol/L insulin (for D) or 25 μmol/L forskolin (for E) for 24 h, followed by measurement of glycogen content. Data are shown as mean ± SD. **P < 0.01 and ***P < 0.001 as comparison between the treated and untreated groups. F: Hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 MOI of Ad-shRNA as indicated. Cells were harvested 72 h later and analyzed by RT-qPCR. The fold change of PPP1R3G mRNA in Ad-Scrambled shRNA group is set to 1. **P < 0.01 in comparison with the Ad-Scrambled shRNA group. G: Cell lysate from hepatocytes of G was used in immunoblotting with the antibodies as indicated. H: Cells as in G were used to measure glycogen content. ***P < 0.001 as compared with the scrambled shRNA group. I: Primary hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 MOI of adenovirus as indicated. Forty-eight hours later, the cells were treated at various glucose concentrations for 24 h, followed by measurement of glycogen content. Data are shown as mean ± SD. *P < 0.05 and **P < 0.01 compared between the two groups. FIG. 3. PPP1R3G stimulates glycogen synthesis in hepatocytes. A: Primary hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 multiplicity of infection (MOI) of Ad-GFP or Ad-PPP1R3G with a Flag tag at the N-terminus. Twenty-four hours later, the cells were harvested for immunoblotting with antibodies as indicated. B: Primary hepatocytes isolated from overnight fasted mice were infected with Ad-GFP or Ad-PPP1R3G at different MOI, followed by glycogen measurement. ***P < 0.001 in comparison with the Ad-GFP group. C: The cells as in A were treated for 24 h with various glucose concentrations, followed by measurement of glycogen content. Data are shown as mean ± SD. **P < 0.01 as comparison between the two groups. D and E: The cells as in A were treated with 100 nmol/L insulin (for D) or 25 μmol/L forskolin (for E) for 24 h, followed by measurement of glycogen content. Data are shown as mean ± SD. **P < 0.01 and ***P < 0.001 as comparison between the treated and untreated groups. F: Hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 MOI of Ad-shRNA as indicated. Cells were harvested 72 h later and analyzed by RT-qPCR. The fold change of PPP1R3G mRNA in Ad-Scrambled shRNA group is set to 1. **P < 0.01 in comparison with the Ad-Scrambled shRNA group. G: Cell lysate from hepatocytes of G was used in immunoblotting with the antibodies as indicated. H: Cells as in G were used to measure glycogen content. ***P < 0.001 as compared with the scrambled shRNA group. I: Primary hepatocytes isolated from overnight-fasted mice were cultured overnight and then treated with 25 MOI of adenovirus as indicated. Forty-eight hours later, the cells were treated at various glucose concentrations for 24 h, followed by measurement of glycogen content. Data are shown as mean ± SD. *P < 0.05 and **P < 0.01 compared between the two groups. More
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GDF11 improves EPC tube formation and migration in vitro. <em>A</em>...
Published: 19 July 2018
Figure 7 GDF11 improves EPC tube formation and migration in vitro. A: Representative images of tube formation in various treatment groups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 12-h incubation with HG. Scale bars = 100 μm. B: Representative images of migration assay in various treatment groups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 24-h incubation with HG. Scale bars = 200 μm. C and D: Quantitative analysis of A and B, respectively. Data are mean ± SEM for five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. hpf, high-power field; MOI, multiplicity of infection. Figure 7. GDF11 improves EPC tube formation and migration in vitro. A: Representative images of tube formation in various treatment groups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 12-h incubation with HG. Scale bars = 100 μm. B: Representative images of migration assay in various treatment groups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1α and then incubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 24-h incubation with HG. Scale bars = 200 μm. C and D: Quantitative analysis of A and B, respectively. Data are mean ± SEM for five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. hpf, high-power field; MOI, multiplicity of infection. More
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Induction of SHP by AMPK inhibits GH-induced PDK4 expression in primary hep...
Published: 13 September 2012
FIG. 3. Induction of SHP by AMPK inhibits GH-induced PDK4 expression in primary hepatocytes. A: Rat primary hepatocytes were infected with 30, 60 multiplicity of infection (MOI) of adenoviral vector expressing CA-AMPK for 36 h. After infection with Ad-CA-AMPK, cells were treated with GH (500 ng/mL) for 1 h. Total RNA extracts were isolated and analyzed by RT-PCR analysis with the indicated primers and then normalized to an internal control (β-actin level). *P < 0.05 and **P < 0.05 compared with untreated control, GH-treated cells. B: Rat primary hepatocytes were infected with Ad-DN-AMPK for 36 h and then treated with GH (500 ng/mL) for 1 h in the presence or absence of metformin (Met) for 12 h. Total RNA were isolated from hepatocytes and used by RT-PCR analysis. SHP, PDK2, and PDK4 mRNA levels were normalized to an internal control with β-actin level. *P < 0.05 and **P < 0.01 compared with untreated control, GH-treated cells. Rat primary hepatocytes were infected with Ad-CA-AMPK (C) and Ad-DN-AMPK (D) for 30 or 60 MOI for 36 h. After infection, cells were treated with GH (500 ng/mL) for 1 h with or without metformin for 12 h. Specific proteins were determined by Western blot analysis with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). E: HepG2 cells were cotransfected with CA-AMPK and DN-AMPK and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. F: Cells were cotransfected with TEL-JAK2, STAT5, CA-AMPK, and DN-AMPK and then treated with metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. All data are representative of at least three independent experiments. All data are shown as fold activations relative to the control (± SEM). FIG. 3. Induction of SHP by AMPK inhibits GH-induced PDK4 expression in primary hepatocytes. A: Rat primary hepatocytes were infected with 30, 60 multiplicity of infection (MOI) of adenoviral vector expressing CA-AMPK for 36 h. After infection with Ad-CA-AMPK, cells were treated with GH (500 ng/mL) for 1 h. Total RNA extracts were isolated and analyzed by RT-PCR analysis with the indicated primers and then normalized to an internal control (β-actin level). *P < 0.05 and **P < 0.05 compared with untreated control, GH-treated cells. B: Rat primary hepatocytes were infected with Ad-DN-AMPK for 36 h and then treated with GH (500 ng/mL) for 1 h in the presence or absence of metformin (Met) for 12 h. Total RNA were isolated from hepatocytes and used by RT-PCR analysis. SHP, PDK2, and PDK4 mRNA levels were normalized to an internal control with β-actin level. *P < 0.05 and **P < 0.01 compared with untreated control, GH-treated cells. Rat primary hepatocytes were infected with Ad-CA-AMPK (C) and Ad-DN-AMPK (D) for 30 or 60 MOI for 36 h. After infection, cells were treated with GH (500 ng/mL) for 1 h with or without metformin for 12 h. Specific proteins were determined by Western blot analysis with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). E: HepG2 cells were cotransfected with CA-AMPK and DN-AMPK and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. F: Cells were cotransfected with TEL-JAK2, STAT5, CA-AMPK, and DN-AMPK and then treated with metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. All data are representative of at least three independent experiments. All data are shown as fold activations relative to the control (± SEM). More
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Metformin-induced upregulation of SHP gene expression is mediated by AMPK. ...
Published: 01 February 2008
FIG. 2. Metformin-induced upregulation of SHP gene expression is mediated by AMPK. A: Rat primary hepatocytes were infected with Ad-AMPK at a multiplicity of infection (MOI) of 30 or 60 for 24 h. Cells were infected with Ad-DN-AMPK for 24 h after 12 h of metformin treatment (2 mmol/l). SHP expression was determined via Northern blot analysis. SHP expression was normalized to GAPDH as an internal control. The data presented are representative of three independently performed Northern blot analyses. B and C: HepG2 and AML12 cell lines were transiently transfected with the indicated reporter genes and treated with the AMPK activator AICAR or the AMPK inhibitor compound C in the presence or absence of metformin for 12 h after transfection. The cells were cotransfected with CA-AMPK, DN-AMPK in the presence or absence of metformin for 12 h after transfection. Luciferase activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. The data presented are representative means from five independent experiments. The data are expressed as fold activation relative to the control (±SEM). Com C, treated with compound C; Met, treated with metformin. FIG. 2. Metformin-induced upregulation of SHP gene expression is mediated by AMPK. A: Rat primary hepatocytes were infected with Ad-AMPK at a multiplicity of infection (MOI) of 30 or 60 for 24 h. Cells were infected with Ad-DN-AMPK for 24 h after 12 h of metformin treatment (2 mmol/l). SHP expression was determined via Northern blot analysis. SHP expression was normalized to GAPDH as an internal control. The data presented are representative of three independently performed Northern blot analyses. B and C: HepG2 and AML12 cell lines were transiently transfected with the indicated reporter genes and treated with the AMPK activator AICAR or the AMPK inhibitor compound C in the presence or absence of metformin for 12 h after transfection. The cells were cotransfected with CA-AMPK, DN-AMPK in the presence or absence of metformin for 12 h after transfection. Luciferase activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. The data presented are representative means from five independent experiments. The data are expressed as fold activation relative to the control (±SEM). Com C, treated with compound C; Met, treated with metformin. More
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Metformin inhibits GH-induced STAT5 transactivity and PDK4 expression in pr...
Published: 13 September 2012
FIG. 2. Metformin inhibits GH-induced STAT5 transactivity and PDK4 expression in primary hepatocytes. Rat primary hepatocytes were cultured under serum-free conditions for 24 h. A: After pretreatment with metformin (Met) for 12 h, the cells were treated with GH for 1 h at the indicated dose (500 ng/mL). B: Rat primary hepatocytes were infected with Ad-SHP at a multiplicity of infection (MOI) of 30 or 60 for 36 h. After infection for 36 h, cells were treated with GH (500 ng/mL) for 1 h. Specific proteins were assayed in whole-cell extracts by Western blot analysis with the indicated antibodies and then normalized to an internal control (β-actin level and/or total forms). C: Rat primary hepatocytes were infected with 60 MOI of Ad-siSHP and Ad-Scram for 36 h. After infection with Ad-siSHP and Ad-Scram, cells were treated with GH (500 ng/mL) for 1 h, with or without metformin for 12 h at the indicated dose. Whole-cell extracts were isolated and analyzed by immunoblotting with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). The results shown are representative of at least three independent experiments. D: HepG2 cell lines were cotransfected with SHP in the indicated reporter genes and treated with GH for 1 h or metformin for 12 h after transfection. Luciferase (Luc) activity was measured after 36 h and normalized to β-galactosidase activity. All data are representative of three independently performed experiments and are shown as fold activations relative to the control (± SEM). E and F: HepG2 cell lines were transfected with the oligonucleotide siSHP and siScram. After transfection for 36 h, cells were transfected with the indicated reporter gene (STAT5-Luc [E], mPDK4-Luc [F]) and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity. The results shown are representative of at least three independent experiments. All data are indicated as fold activations relative to the control (± SEM). FIG. 2. Metformin inhibits GH-induced STAT5 transactivity and PDK4 expression in primary hepatocytes. Rat primary hepatocytes were cultured under serum-free conditions for 24 h. A: After pretreatment with metformin (Met) for 12 h, the cells were treated with GH for 1 h at the indicated dose (500 ng/mL). B: Rat primary hepatocytes were infected with Ad-SHP at a multiplicity of infection (MOI) of 30 or 60 for 36 h. After infection for 36 h, cells were treated with GH (500 ng/mL) for 1 h. Specific proteins were assayed in whole-cell extracts by Western blot analysis with the indicated antibodies and then normalized to an internal control (β-actin level and/or total forms). C: Rat primary hepatocytes were infected with 60 MOI of Ad-siSHP and Ad-Scram for 36 h. After infection with Ad-siSHP and Ad-Scram, cells were treated with GH (500 ng/mL) for 1 h, with or without metformin for 12 h at the indicated dose. Whole-cell extracts were isolated and analyzed by immunoblotting with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). The results shown are representative of at least three independent experiments. D: HepG2 cell lines were cotransfected with SHP in the indicated reporter genes and treated with GH for 1 h or metformin for 12 h after transfection. Luciferase (Luc) activity was measured after 36 h and normalized to β-galactosidase activity. All data are representative of three independently performed experiments and are shown as fold activations relative to the control (± SEM). E and F: HepG2 cell lines were transfected with the oligonucleotide siSHP and siScram. After transfection for 36 h, cells were transfected with the indicated reporter gene (STAT5-Luc [E], mPDK4-Luc [F]) and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity. The results shown are representative of at least three independent experiments. All data are indicated as fold activations relative to the control (± SEM). More
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dSA triggers senescence in Ins-1 cells. <em>A</em>: Quantification ...
Published: 13 March 2014
Figure 2 dSA triggers senescence in Ins-1 cells. A: Quantification of β-gal activity following incubation for 24 h with 1 μmol/L SA, 0.5 and 1 μmol/L dSA, or BSA as control. Values are normalized to the number of live cells. B: Immunofluorescence imaging showing nuclear expression of p21 following incubation for 24 h with 1 μmol/L SA, 1 μmol/L dSA, or BSA as control. Nuclei are stained with DAPI (blue). The lower panels show nuclear localization of p21. C: Western blot and densitometric quantification of p21/GAPDH levels following incubation for 24 h with 1 μmol/L SA, 1 μmol/L dSA, or BSA as control. D: Immunostaining of p21 (upper panels) or live imaging (lower panels) of Ins-1 cells infected with Adp21 and AdGFP at MOI50. E: Enumeration of live Ins-1 cells 24 h after Adp21 or AdGFP infection. Note how Adp21 decreased the replication of Ins-1 cells in a dose–response manner without inducing cell death. F: Quantification of β-gal activity 24 h after Adp21 or AdGFP infection. Note how senescence is induced only in the presence of Adp21. Results are mean ± SEM (n = 3). Scale bars: 50 μm. *P < 0.05. β-gal, β-galactosidase; cntl, control; dSA, 1-deoxysphinganine; MOI, multiplicity of infection; SA, sphinganine. Figure 2. dSA triggers senescence in Ins-1 cells. A: Quantification of β-gal activity following incubation for 24 h with 1 μmol/L SA, 0.5 and 1 μmol/L dSA, or BSA as control. Values are normalized to the number of live cells. B: Immunofluorescence imaging showing nuclear expression of p21 following incubation for 24 h with 1 μmol/L SA, 1 μmol/L dSA, or BSA as control. Nuclei are stained with DAPI (blue). The lower panels show nuclear localization of p21. C: Western blot and densitometric quantification of p21/GAPDH levels following incubation for 24 h with 1 μmol/L SA, 1 μmol/L dSA, or BSA as control. D: Immunostaining of p21 (upper panels) or live imaging (lower panels) of Ins-1 cells infected with Adp21 and AdGFP at MOI50. E: Enumeration of live Ins-1 cells 24 h after Adp21 or AdGFP infection. Note how Adp21 decreased the replication of Ins-1 cells in a dose–response manner without inducing cell death. F: Quantification of β-gal activity 24 h after Adp21 or AdGFP infection. Note how senescence is induced only in the presence of Adp21. Results are mean ± SEM (n = 3). Scale bars: 50 μm. *P < 0.05. β-gal, β-galactosidase; cntl, control; dSA, 1-deoxysphinganine; MOI, multiplicity of infection; SA, sphinganine. More
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Role of KLF15 in metformin-induced suppression of the genes for gluconeogen...
Published: 14 April 2010
FIG. 3. Role of KLF15 in metformin-induced suppression of the genes for gluconeogenic or amino acid–degrading enzymes. A: HL1c cells were incubated for 6 h in the absence or presence of 100 μmol/l 8-CPT-cAMP and 500 nmol/l dexamethasone (Dex/cAMP) as well as with the indicated concentrations of metformin (Met). The amounts of KLF15 (upper panel) and of KLF15, HPD, PEPCK, and G6Pase mRNAs (lower panels) were then determined by immunoblot (IB) and RT and real-time PCR analyses, respectively. B and C: HL1c cells that had been infected with an adenoviral vector encoding Flag epitope–tagged KLF15 at the indicated multiplicity of infection (MOI) (plaque-forming units per cell) were incubated for 6 h with 500 nmol/l dexamethasone in the absence or presence of 5 mmol/l metformin. The cells were then subjected to immunoblot (IB) analysis with antibodies specific for KLF15 or for α-tubulin (loading control) (B) or to RT and real-time PCR analysis of HPD and PEPCK mRNAs (C). All RT-PCR data are means ± SEM from three or four independent experiments. **P < 0.01. Immunoblots are representative of at least three experiments. FIG. 3. Role of KLF15 in metformin-induced suppression of the genes for gluconeogenic or amino acid–degrading enzymes. A: HL1c cells were incubated for 6 h in the absence or presence of 100 μmol/l 8-CPT-cAMP and 500 nmol/l dexamethasone (Dex/cAMP) as well as with the indicated concentrations of metformin (Met). The amounts of KLF15 (upper panel) and of KLF15, HPD, PEPCK, and G6Pase mRNAs (lower panels) were then determined by immunoblot (IB) and RT and real-time PCR analyses, respectively. B and C: HL1c cells that had been infected with an adenoviral vector encoding Flag epitope–tagged KLF15 at the indicated multiplicity of infection (MOI) (plaque-forming units per cell) were incubated for 6 h with 500 nmol/l dexamethasone in the absence or presence of 5 mmol/l metformin. The cells were then subjected to immunoblot (IB) analysis with antibodies specific for KLF15 or for α-tubulin (loading control) (B) or to RT and real-time PCR analysis of HPD and PEPCK mRNAs (C). All RT-PCR data are means ± SEM from three or four independent experiments. **P < 0.01. Immunoblots are representative of at least three experiments. More
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Dose-related, Dox-induced expression of cdk6 or cyclin D<sub>1</sub> from A...
Published: 17 January 2012
FIG. 1. Dose-related, Dox-induced expression of cdk6 or cyclin D1 from Ad.TRE in human islets. A and B: Immunoblots of induced cdk6 or cyclin D1 expression, respectively, in intact human islets 72 h posttransduction with adenoviruses as indicated, and treated with or without increasing doses of Dox. Delivery of cdk6 and cyclin D1 was confirmed using the HA epitope tag. C and D: Densitometric quantification of A and B (n = 5). Values are expressed as a percentage of the Dox 0.1 group. P values refer to significance of indicated bars as compared with Ad.GFP-Ad.TTA controls as determined by Student unpaired t test. Error bars indicate SEM. Human islet donor information was as follows: mean age 43.7 ± 2.1 years; gender 16 men, 17 women; BMI 27.5 ± 0.9; purity 68.7 ± 3.5%. Islets were maintained in RPMI 1640 medium containing 5.5 mmol/L glucose, 10% tet-free FBS (Clontech, Mountain View, CA), and 1% penicillin/streptomycin, unless otherwise stated. Two types of adenoviruses were used in this tet-on system. In the first, the TTA is driven by the CMV promoter. In the second, the cDNAs encoding either human cyclin D1 or human cdk6 were controlled by the TRE. Both the cdk6 and cyclin D1 constructs contained a HA epitope tag. The adenoviruses were generated using pJM17 ( 8 , 9 ) and a modified pAC-CMVpLpA ( 8 , 9 ). In the Ad.TRE-cdk6 and Ad.TRE-cyclin D1 viruses, the CMV promoter in the pAC-CMVpLpA plasmid was replaced with the MfeI-EcoRI promoter fragment of the pTRE-Shuttle2 from the Tet-On System (Clontech), now referred to as pAC-TRE, and the cdk6-HA and HA-cyclin D1 cDNAs were ligated into pAC-TRE. Similarly, the cDNA for the reverse TTA from pTet-On Advanced (Clontech) was subcloned into pACCMVpLpA to produce the TTA adenovirus. Intact islets were transduced with adenovirus at a total of 250 multiplicities of infection (MOI) (150 MOI of Ad.TRE/100 MOI of Ad.TTA) in RPMI 1640 medium for 1 h. In some experiments, islets were dispersed by trypsinizing for 10 min at 37°C. Complete medium was added to stop the digestion. These single dispersed human islet cells were plated on 12-mm glass coverslips in 24-well plates and transduced at 150 MOI (50 MOI of each virus) in complete media for 2 h. Dox was added to the media immediately after transduction and replaced every other day as depicted. (A high-quality color representation of this figure is available in the online issue.) FIG. 1. Dose-related, Dox-induced expression of cdk6 or cyclin D1 from Ad.TRE in human islets. A and B: Immunoblots of induced cdk6 or cyclin D1 expression, respectively, in intact human islets 72 h posttransduction with adenoviruses as indicated, and treated with or without increasing doses of Dox. Delivery of cdk6 and cyclin D1 was confirmed using the HA epitope tag. C and D: Densitometric quantification of A and B (n = 5). Values are expressed as a percentage of the Dox 0.1 group. P values refer to significance of indicated bars as compared with Ad.GFP-Ad.TTA controls as determined by Student unpaired t test. Error bars indicate SEM. Human islet donor information was as follows: mean age 43.7 ± 2.1 years; gender 16 men, 17 women; BMI 27.5 ± 0.9; purity 68.7 ± 3.5%. Islets were maintained in RPMI 1640 medium containing 5.5 mmol/L glucose, 10% tet-free FBS (Clontech, Mountain View, CA), and 1% penicillin/streptomycin, unless otherwise stated. Two types of adenoviruses were used in this tet-on system. In the first, the TTA is driven by the CMV promoter. In the second, the cDNAs encoding either human cyclin D1 or human cdk6 were controlled by the TRE. Both the cdk6 and cyclin D1 constructs contained a HA epitope tag. The adenoviruses were generated using pJM17 (8,9) and a modified pAC-CMVpLpA (8,9). In the Ad.TRE-cdk6 and Ad.TRE-cyclin D1 viruses, the CMV promoter in the pAC-CMVpLpA plasmid was replaced with the MfeI-EcoRI promoter fragment of the pTRE-Shuttle2 from the Tet-On System (Clontech), now referred to as pAC-TRE, and the cdk6-HA and HA-cyclin D1 cDNAs were ligated into pAC-TRE. Similarly, the cDNA for the reverse TTA from pTet-On Advanced (Clontech) was subcloned into pACCMVpLpA to produce the TTA adenovirus. Intact islets were transduced with adenovirus at a total of 250 multiplicities of infection (MOI) (150 MOI of Ad.TRE/100 MOI of Ad.TTA) in RPMI 1640 medium for 1 h. In some experiments, islets were dispersed by trypsinizing for 10 min at 37°C. Complete medium was added to stop the digestion. These single dispersed human islet cells were plated on 12-mm glass coverslips in 24-well plates and transduced at 150 MOI (50 MOI of each virus) in complete media for 2 h. Dox was added to the media immediately after transduction and replaced every other day as depicted. (A high-quality color representation of this figure is available in the online issue.) More
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<em>Sox4</em> upregulates SREBP-1c expression. <em>A</em>: ...
Published: 04 September 2018
Figure 4 Sox4 upregulates SREBP-1c expression. A: Representative protein levels of SOX4 in Hep1-6 cells transfected with Ad-Sox4 or Ad-GFP. B and C: Cellular TG content (B) and gene expression (C) in Hep1-6 cells with Sox4 overexpression. D: Relative mRNA levels of Sox4 in Hep1-6 cells transfected with two siRNA oligos targeting Sox4. E and F: Cellular TG content (E) and gene expression (F) in Hep1-6 cells with Sox4 deficiency. G and H: Cellular TG contents and relative mRNA levels of SREBP-1c in Hep1-6 cells transfected with two independent Sox4 siRNA oligos or negative control in the presence of low (5 mmol/L) or high (25 mmol/L) levels of glucose. I and J: Cellular TG content and relative mRNA levels of SREBP-1c in Hep1-6 cells transfected with two independent Sox4 siRNA oligos or negative control in the presence of BSA or palmitate. K: Pearson r and P values for normalized Sox4 mRNA levels versus SREBP-1c levels in human livers (n = 32). MOI, multiplicity of infection; NC, negative control. Statistical significance is displayed as *P < 0.05, **P < 0.01, or ***P < 0.001. Figure 4. Sox4 upregulates SREBP-1c expression. A: Representative protein levels of SOX4 in Hep1-6 cells transfected with Ad-Sox4 or Ad-GFP. B and C: Cellular TG content (B) and gene expression (C) in Hep1-6 cells with Sox4 overexpression. D: Relative mRNA levels of Sox4 in Hep1-6 cells transfected with two siRNA oligos targeting Sox4. E and F: Cellular TG content (E) and gene expression (F) in Hep1-6 cells with Sox4 deficiency. G and H: Cellular TG contents and relative mRNA levels of SREBP-1c in Hep1-6 cells transfected with two independent Sox4 siRNA oligos or negative control in the presence of low (5 mmol/L) or high (25 mmol/L) levels of glucose. I and J: Cellular TG content and relative mRNA levels of SREBP-1c in Hep1-6 cells transfected with two independent Sox4 siRNA oligos or negative control in the presence of BSA or palmitate. K: Pearson r and P values for normalized Sox4 mRNA levels versus SREBP-1c levels in human livers (n = 32). MOI, multiplicity of infection; NC, negative control. Statistical significance is displayed as *P < 0.05, **P < 0.01, or ***P < 0.001. More
Journal Articles
Journal: Diabetes
Diabetes 2015;64(12):4088–4098
Published: 29 September 2015
... among controls was performed in parallel to virus infections but in the absence of virus. For infection in vitro, PHTTg or HuH7.5.1 cells (1 × 106) were infected with J399EM for the indicated time, with the indicated multiplicities of infection (MOIs). RNA isolation, cDNA...
Includes: Supplementary data
Journal Articles
Journal: Diabetes
Diabetes 1989;38(9):1103–1108
Published: 01 September 1989
... plaques and no detectable levels of IFN (8). added to a 12-well tissue-culture plate. Most (85-90%) of Each virus was diluted in Hanks' balanced salt solution the cells were identified as (3-cells by fluorescein isothio- (HBSS) to give the indicated multiplicity of infection (MOI). cyanate-labeled anti...