Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1/AMPK-α/NF-κB pathway in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + SIRT1-siRNA for 24 h, followed by OGD/R treatment. Then, the (A) protein and (B) mRNA expression levels of SIRT1 were determined by western blot and reverse transcription-quantitative PCR analyses, respectively. Additionally, the protein levels of p-AMPK-α, AMPK-α, p65, and p-p65 were measured by western blotting. The ratios of (C) p-AMPK-α/AMPK-α and (D) p-p65/p65 normalized to β-actin were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; SIRT1, sirtuin 1; p, phosphorylated; AMPK-α, AMP-activated protein kinase-α.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1/AMPK-α/NF-κB pathway in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + SIRT1-siRNA for 24 h, followed by OGD/R treatment. Then, the (A) protein and (B) mRNA expression levels of SIRT1 were determined by western blot and reverse transcription-quantitative PCR analyses, respectively. Additionally, the protein levels of p-AMPK-α, AMPK-α, p65, and p-p65 were measured by western blotting. The ratios of (C) p-AMPK-α/AMPK-α and (D) p-p65/p65 normalized to β-actin were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; SIRT1, sirtuin 1; p, phosphorylated; AMPK-α, AMP-activated protein kinase-α.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: SIRT1 is a direct target of miR-217. (A) Interaction between miR-217 and the 3′-UTR of SIRT1 predicted using TargetScan. (B) Level of miR-217 in neurons transfected with miR-217 mimic or mimic control as determined by RT-qPCR. (C) Luciferase activity of reporters containing the WT or MUT SIRT1 3′-UTR following transfection with miR-217 mimic or mimic control. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. mimic control. (D) Protein and (E) mRNA expression of SIRT1 in neurons following OGD/R treatment as determined by western blot and RT-qPCR analyses, respectively. Data are presented as the mean ± standard deviation. **P<0.01 vs. Control. RT-qPCR, reverse transcription-quantitative PCR; SIRT1, sirtuin 1; 3′-UTR, 3′-untranslated region; miR-217, microRNA-217; WT, wild-type; MUT, mutated; OGD/R, oxygen-glucose deprivation and reoxygenation.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Standard Deviation, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1 expression in neurons. (A) Relative miR-217 expression in neurons following transfection with miR-217 inhibitor or inhibitor control. Relative SIRT1 mRNA expression in neurons following (B) siRNA and/or (C) inhibitor transfection. Protein level of SIRT1 in neurons following (D) siRNA and/or (E) inhibitor transfection. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. inhibitor. miR-217, microRNA-217; SIRT1, sirtuin 1; siRNA, small interfering RNA.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Expressing, Transfection, Control, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on OGD/R-induced neuronal injury. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + sirtuin 1-siRNA for 24 h, followed by OGD/R treatment. (A) Cell viability was detected using a Cell Counting Kit-8 assay. (B) LDH release was measured by an LDH assay. (C and D) Cell apoptosis was determined using flow cytometry. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. control; # P<0.05, ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; LDH, lactate dehydrogenase; siRNA, small interfering RNA; PI, propidium iodide.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Cell Counting, Lactate Dehydrogenase Assay, Flow Cytometry, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on proinflammatory factor levels in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + sirtuin 1-siRNA for 24 h, followed by OGD/R treatment. Then, the levels of (A) TNF-α, (B) IL-6 (C) and IL-1β in the supernatant of neurons was measured using ELISA. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; && P<0.01 vs. inhibitor. miR-217, microRNA-217; TNF-α, tumor necrosis factor-α; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; IL, interleukin.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on oxidative stress in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + sirtuin 1-siRNA for 24 h, followed by OGD/R treatment. Then, (A) ROS, (B) MDA and (C) SOD levels were determined. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. control; # P<0.05, ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; ROS, reactive oxygen species; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; MDA, malondialdehyde; SOD, superoxide dismutase.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1/AMPK-α/NF-κB pathway in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + SIRT1-siRNA for 24 h, followed by OGD/R treatment. Then, the (A) protein and (B) mRNA expression levels of SIRT1 were determined by western blot and reverse transcription-quantitative PCR analyses, respectively. Additionally, the protein levels of p-AMPK-α, AMPK-α, p65, and p-p65 were measured by western blotting. The ratios of (C) p-AMPK-α/AMPK-α and (D) p-p65/p65 normalized to β-actin were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; SIRT1, sirtuin 1; p, phosphorylated; AMPK-α, AMP-activated protein kinase-α.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1/AMPK-α/NF-κB pathway in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + SIRT1-siRNA for 24 h, followed by OGD/R treatment. Then, the (A) protein and (B) mRNA expression levels of SIRT1 were determined by western blot and reverse transcription-quantitative PCR analyses, respectively. Additionally, the protein levels of p-AMPK-α, AMPK-α, p65, and p-p65 were measured by western blotting. The ratios of (C) p-AMPK-α/AMPK-α and (D) p-p65/p65 normalized to β-actin were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; SIRT1, sirtuin 1; p, phosphorylated; AMPK-α, AMP-activated protein kinase-α.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1

doi: 10.3892/mmr.2019.10317

Figure Lengend Snippet: Effects of miR-217 inhibitor on SIRT1/AMPK-α/NF-κB pathway in OGD/R-treated neurons. Neurons were transfected with miR-217 inhibitor, inhibitor control or miR-217 inhibitor + SIRT1-siRNA for 24 h, followed by OGD/R treatment. Then, the (A) protein and (B) mRNA expression levels of SIRT1 were determined by western blot and reverse transcription-quantitative PCR analyses, respectively. Additionally, the protein levels of p-AMPK-α, AMPK-α, p65, and p-p65 were measured by western blotting. The ratios of (C) p-AMPK-α/AMPK-α and (D) p-p65/p65 normalized to β-actin were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. control; ## P<0.01 vs. OGD/R; & P<0.05, && P<0.01 vs. inhibitor. miR-217, microRNA-217; OGD/R, oxygen-glucose deprivation and reoxygenation; siRNA, small interfering RNA; SIRT1, sirtuin 1; p, phosphorylated; AMPK-α, AMP-activated protein kinase-α.

Article Snippet: Equal amounts of protein samples (25 μg/lane) were separated via SDS-PAGE on 12% gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), and blocked in 5% skim milk at room temperature for 1.5 h. Then, the membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), phosphorylated (p)-AMP-activated protein kinase-α (AMPK-α; 1:1,000; cat. no. 50081; Cell Signaling Technology, Inc.), AMPK-α (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein blots were visualized using chemiluminescent ECL reagent (EMD Millipore) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Control, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA