Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 1. ALA activates SIRT1/LKB1 pathway in HepG2 cells. (A) The dose–response effect of ALA on SIRT1 deacetylase activity. HepG2 cells were treated with 0, 50, 125, 250 and 500 μM ALA, or 0, 10, 25, 50 and 100 μM resveratrol used as a positive control for 24 h. Data are presented as mean±S.E.M. (n=5). *Pb.05, **Pb.01 vs. control (0 μM ALA); #Pb.05, ##Pb.01 vs. control (0 μM resveratrol). (B) Intracellular NAD+/NADH ratio. HepG2 cells were treated with 0 (control), 250, 500 and 1000 μM ALA for 24 h. Data are presented as mean±S.E.M. (n=6). *Pb.05, **Pb.01 vs. control (0 μmol ALA). HepG2 cells were transfected with SIRT1siRNA or scramble siRNA for 24 h after incubation with ALA (250 μM, 6 h). (C) IP of acetylated liver kinase B1 (LKB1). *Pb.05 vs. control (untreated HepG2 cells); #Pb.05 vs. ALA group; ΔPb.05 vs. ALA+scramble siRNA group. (D) COIP of SIRT1 and LKB1. Nonspecific IgG was used as control.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Histone Deacetylase Assay, Activity Assay, Positive Control, Control, Transfection, Incubation

Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 3. ALA causes redistribution of transcription factors FoxO1 and SREBP-1 via the SIRT1/LKB1/AMPK signaling pathway. (A and B) HepG2 cells were treated with 125 μM PA and 250 μM ALA for 12 h. Starvation: HepG2 was incubated with serum-free medium. FoxO1 and SREBP-1 protein expression and distribution were determined by immunofluorescence staining (magnification, 400×). Green: FITC; blue: DAPI. (C) HepG2 cells were treated with 0 (control, C), 50, 125, 250, 500 and 1000 μM ALA for 12 h. *Pb.05, #Pb.05 vs. control (untreated cells). (D) Effect of ALA (250 μM, 6 h) on FoxO1 and SREBP-1 phosphorylation levels in the presence or absence of AMPK inhibitor (CC, 20 μM, 0.5 h), SIRT1 inhibitor (NA, 10 mM, 24 h) and AMPK activator (AICAR, 2 mM, 1 h), respectively. *Pb.05, #Pb.05 vs. ALA group.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Incubation, Expressing, Immunofluorescence, Staining, Control, Phospho-proteomics

Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 6. Proposed scheme illustrating the role of ALA in the regulation of hepatocyte lipid metabolism and antioxidation. ALA reverses the HFD-induced changes in AMPK expression, which is involved in SIRT1/LKB1/AMPK-mediated signaling, and transcrip- tion factors FoxO1, SREBP-1 and nuclear Nrf2 protein expression, along with changes in the lipid metabolism and antioxidative enzyme expression.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Expressing

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 1. Gene and protein expression of LKB1 in GC muscles of all experimental groups. (A-D) RT-PCR and western blot (WB) analyses of LKB1 expression in the following mice: (A) C57BL/10 mdx mice (n=10) versus C57BL/10 WT mice (n=11) (*P<0.036); (B) D2 mdx mice (n=12) versus D2 WT mice (n=9) (*P<0.0192); (C) C57BL/10 mdx exercised (EXER) mice (n=4) versus C57BL/10 mdx sedentary (SED) mice (n=4) (*P=0.0153); (D) C57BL/10 mdx mice treated with metformin (MET) (n=4) versus C57BL/10 mdx mice (n=4) (*P=0.015). Blots were loaded by an operator unaware of the experimental groupings of samples. In A, the most representative blot of the three western blot experiments performed is shown because of the large number of samples belonging to Group 1. Data are mean+s.e.m. Significant differences were assessed by unpaired Student’s t-test.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Expressing, Muscles, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 2. Immunofluorescence imaging for LKB1. GC muscle sections of 6-month-old BL10 mdx and WT mice were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown at the bottom.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 7. Gene expression and localization of LKB1 in WT and dystrophic murine muscle cells. (A) Lkb1 expression in 2B4 (WT) and SF1 (DMD) myoblasts (D0), and myocytes (D2-D11). Significant differences were assessed by unpaired Student’s t-test (*P≤0.05; ***P≤0.001). ns, not significant. (B) Immunofluorescence imaging for LKB1. D11 2B4 and SF1 cells were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown on the right.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Gene Expression, Expressing, Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 8. Schematic summarizing the pathways involved in the LKB1- AMPK-HDAC axis in healthy and dystrophic muscle. In healthy muscle, LKB1 is activated in response to exercise, in turn stimulating the metabolic remodeling mediated by the key modulator AMPK. AMPK turns off the class II HDACs that are also inhibited via SIK phosphorylation by LKB1. In dystrophic muscle, this refined mechanism of regulation is altered owing to LKB1 impairment. This allows class II HDACs to carry on their epigenetic silencer action, thus inhibiting the expression of genes involved in oxidative metabolism (Mef2c).

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Phospho-proteomics, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Thymoquinone‑induced autophagy mitigates doxorubicin‑induced H9c2 cell apoptosis

doi: 10.3892/etm.2022.11630

Figure Lengend Snippet: TQ activates the LKB1/AMPK/ULK1 pathway. (A) Western blot analysis shows the expression of p-LKB1, LKB1, p-AMPKα, AMPKα, p-ULK1 (ser317), p-mTOR, and p-ULK1 (ser575) after Dox or Dox in combination use with TQ. (B) Relative intensity (% of loading control) of p-LKB1, LKB1, p-AMPKα, AMPKα, p-ULK1 (ser317), p-mTOR, and p-ULK1 (ser575) after Dox or Dox in combination use with TQ which was determined by Image J software. (C) Relative intensity (% of total subtype) of p-LKB1, LKB1, p-AMPKα, AMPKα, p-ULK1 (ser317), p-mTOR, and p-ULK1 (ser575) after Dox or Dox in combination use with TQ which was determined by Image J software. Data are presented as the mean ± SD of three independent experiments. A one-way ANOVA test with post hoc Student-Newman-Keuls test was used for evaluating the difference. Significance was indicated at P<0.05: *P<0.05, Dox vs. the Dox+TQ group. TQ, thymoquinone; Dox, doxorubicin; LKB1, liver kinase B1; AMPK, AMP-activated protein kinase; ULK1, unc-51-like kinase 1; mTOR, mammalian target of rapamycin; p-, phosphorylated.

Article Snippet: Rabbit-cleaved caspase-3 (cat: 9664), LC3B (cat: 3868), phosphorylated (p-)LKB1 (cat: 3050), LKB1 (cat: 3055), AMPKα (cat: 5832), p-AMPKα (Thr172) (cat: 50081), p-ULK1 (Ser555) (cat: 5869), p-ULK1 (Ser317) (cat: 37762), ULK1 (cat: 8054), mTor (cat: 2972), p-mTOR (cat: 5536), and β-actin (cat: 3700) (dilution 1:1,000, Cell Signaling Technology, Inc.), were incubated with the membranes at 4 ̊C overnight, which were further incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (cat: 7074) (dilution 1:2,000; Cell Signaling Technology, Inc.) for 1 h at RT.

Techniques: Western Blot, Expressing, Control, Software

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Reverse-transcription quantitative polymerase chain reaction primers used in the present study.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: List of primary antibodies used in the present study.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Isoflurane activates Lkb1-p21 signalling in WT NSCs, although not in NE-4C (p53 −/− ) cells. (A) RT-qPCR analysis reveals that the mRNA expression levels of Lkb1 , p53 and p21 in WT NSCs treated with an IC 50 concentration of isoflurane were significantly higher compared with that in untreated cells ( ** P<0.01; # P>0.05 compared with the non-treated group; n=3). (B) RT-qPCR analysis indicates that the mRNA expression levels of p53 and p21 in isoflurane-treated NE-4C (p53 −/− ) cells were unchanged compared with the untreated cells ( ** P<0.01; * P<0.05; # P>0.05 compared with the non-treated group; n=3). (C) Western blotting indicates that the protein expression levels of Lkb1 and p21 in isoflurane-treated WT NSCs were significantly increased compared with untreated cells ( ** P<0.01; * P<0.05; # P>0.05 compared with the non-treated group; n=3). (D) Western blotting indicates that only the protein expression level of Lkb1 was increased in isoflurane-treated NE-4C (p53 −/− ) cells ( ** P<0.01; # P>0.05 compared with the non-treated group; n=3). GAPDH was used as a loading control. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT NSCs, wild-type neural stem cells.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Immunofluorescence analysis of the Lkb1-p21 signalling pathway in WT NSCs. The protein expression levels of LKB1, p53-Ser-pho and p21 in isoflurane-treated WT NSCs were significantly increased compared with the untreated cells (scale bar, 50 µ m). FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; WT NSCs, wild-type neural stem cells; p53-Ser-pho, Ser15-phosphorylated p53.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Immunofluorescence, Expressing

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Immunofluorescence analysis of the Lkb1-p21 signalling pathway in NE-4C (p53 −/− ) cells. Only Lkb1 expression was increased in isoflurane-treated NE-4C (p53 −/− ) cells compared with the untreated controls (scale bar, 50 µ m). FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; WT NSCs, wild-type neural stem cells; p53-Ser-pho, Ser15-phosphorylated p53.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Immunofluorescence, Expressing