Journal: Disease Markers

Article Title: Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways

doi: 10.1155/2022/7052176

Figure Lengend Snippet: Leptin mediates β -catenin activation through the crosstalk between MTA1/WNT and PI3K/AKT pathways in HTR-8/SVneo cells. (a) HTR-8/SVneo cells were treated with exogenous leptin (0 and 200 ng/ml) for 24 h, and MTA1, WNT1, p-GSK3 β (Ser9), and p-AKT (Ser473) levels were detected by Western blot. Data are shown as mean ± SD; ∗ P < 0.01 vs. leptin (0 ng/ml). (b) The knockdown efficiencies of MTA1 and WNT1 were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (c) HTR-8/SVneo cells were transfected with MTA1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of MTA1, WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (d) HTR-8/SVneo cells were transfected with WNT1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (e) The knockdown efficiencies of AKT and β -catenin were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (f) HTR-8/SVneo cells were transfected with AKT siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of AKT, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. All experiments were performed in triplicate.

Article Snippet: The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3 β (ab32391), β -catenin (ab32572), histone 3 (ab1791), β -actin (ab6276) (Abcam, USA); p-AKT (Ser473) (#4060), p-GSK3 β (Ser9) (#9322) (Cell Signaling Technology, USA).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Transfection, Expressing

Journal: BioMed Research International

Article Title: The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy

doi: 10.1155/2014/961438

Figure Lengend Snippet: Protein expression in GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last 10 days). Relative optical density of total Akt (tAkt) and phosphorylated Akt (P-Akt) ((a) and (c)), total GSK3 β (tGSK3 β ) and phosphorylated GSK3 β (P-GSK3 β ) ((b) and (d)), and tAkt/P-Akt and tGSK3 β /P-GSK3 β ratios ((e) and (f), resp.). Bands representing tAkt, P-Akt, tGSK3, and P-GSK3 β are illustrated in (g). α -Tubulin (55 kDa) was used as control. The numbers represent the mean ± S.E.M. CT = control group ( n = 6), DX = dexamethasone group ( n = 5), n-3 = n-3 PUFA group ( n = 6), and DX + n-3 = dexamethasone + n-3 PUFA group ( n = 6). Values represent means ± S.E.M., analyzed by Student's t -test, # P < 0.05.

Article Snippet: The primary antibodies used were Akt (1 : 2,000, Cell Signaling #4691), P-Akt (1 : 2,000, Cell Signaling #4060, Ser473); mTOR (1 : 500, Cell Signaling #2972), P-mTOR (1 : 500, Cell Signaling #2971, Ser2448), GSK3 β (1 : 2,000, Cell Signaling #9315), P-GSK3 β (1 : 2,000, Cell Signaling #9322, Ser9), FoXO3a (1 : 500, Cell Signaling #2497), P-FoXO3a (1 : 500, Cell Signaling #9466, Ser253), and α -tubulin (1 : 30,000, Hybridoma bank) in 5% BSA/TBS-T.

Techniques: Expressing

Journal: PPAR Research

Article Title: Bezafibrate Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis

doi: 10.1155/2017/5789714

Figure Lengend Snippet: The effect of BZA on AKT/GSK3 β , AMPK α , and MAPK signal pathways. The relative quantitative results of PPAR- α , P-AKT, P-GSK3 β , P-ERK, P-P38, and P-AMPK α in the four groups ( n = 6).

Article Snippet: The following first antibodies were purchased from Cell Signaling Technology: anti-AKT (#4691), anti-phospho-AKT (#4060), anti-GSK3 β (#9315), anti-phospho-GSK3 β (#9323P), anti-ERK (#4695), anti-phospho-ERK (#4370P), anti-P38 (#9212P), anti-phospho-P38 (#4511P), anti-AMPK α (#2603P), and anti-phospho-AMPK α (#2535).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

doi: 10.1155/2018/8597897

Figure Lengend Snippet: Salidroside (SAL) increases phosphorylation of AMPK, ACC, Akt, and GSK3 β and suppresses activation of NLRP3 inflammasome in HFD mice. After treatment with SAL (100 mg·kg −1 ·d −1 ) for 8 weeks, liver tissues were obtained from RD and HFD mice. For the assessment of insulin sensitivity in vivo , mice were stimulated with 0.75 mU·g −1 insulin for 10 min after a 12 h fast before sacrificed. The phosphorylation of AMPK, ACC (a), Akt, and GSK3 β (b) and the activation of NLRP3 inflammasome (c) were analyzed by immunoblot. † P < 0.05, †† P < 0.01 versus RD mice treated with vehicle; ∗ P < 0.05, ∗∗ P < 0.01 versus HFD mice treated with vehicle. Values are means ± s.e.m. ( n = 3).

Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

Techniques: Activation Assay, In Vivo, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

doi: 10.1155/2018/8597897

Figure Lengend Snippet: Salidroside (SAL) improves insulin sensitivity and suppresses NLRP3 inflammasome activation in hepatocytes. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), primary mouse hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, and 10 μ M) for 72 h. For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. Protein sample were extracted from hepatocytes or supernatant (SN). The phosphorylation of Akt and GSK3 β (a, d) and the activation of NLRP3 inflammasome (c, f) were analyzed by immunoblot. The supernatant IL-1 β concentration (b, e) was measured by ELISA method. † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a), (c), (d), and (f): n = 4; (b) and (e): n = 3).

Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

Techniques: Activation Assay, Cell Culture, Incubation, In Vitro, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Salidroside Attenuates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via AMPK-Dependent TXNIP/NLRP3 Pathway

doi: 10.1155/2018/8597897

Figure Lengend Snippet: Salidroside (SAL) protects hepatocytes against ROS overproduction. After cultured overnight in the serum-free medium which contains normal glucose (NG, 5.5 mM), hepatocytes were incubated in the serum-free medium which contains 30 mM glucose and 100 nM insulin (HG) and treated with 10 μ M SAL for the indicated periods of time (0–72 h), or treated with SAL (0.1, 1, 10 μ M) and N-acetylcysteine (NAC, 5 mM) for 72 h. The ROS levels were detected using DCF-DA and MitoSOX as indicated (a–d). The Oil Red O staining (e) and lipid content analysis (f) were performed. Scale bar = 200 μ m. Protein sample was extracted from hepatocytes or supernatant (SN). For assessment of insulin sensitivity in vitro , hepatocytes were treated as indicated, the medium was removed, and cells were incubated in fresh serum-free DMEM containing insulin (10 nM) for 20 min before protein samples were extracted. The phosphorylation of AMPK, ACC (g), Akt, and GSK3 β (h) and the activation of NLRP3 inflammasome (j) were analyzed by immunoblot. The supernatant IL-1 β concentration was measured by ELISA method (i). † P < 0.05, †† P < 0.01 versus NG; ∗ P < 0.05, ∗∗ P < 0.01 versus HG. Values are means ± s.e.m. ((a)–(d), (f)–(h), and (j): n = 4; (e) and (i): n = 3).

Article Snippet: The following primary antibodies were used: anti-IL-1 β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr 172 (number 2535), anti-phospho-GSK3 β Ser 9 (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser 79 (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser 473 (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3 β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA).

Techniques: Cell Culture, Incubation, Staining, In Vitro, Activation Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemether Activation of AMPK/GSK3 β (ser9)/Nrf2 Signaling Confers Neuroprotection towards β- Amyloid-Induced Neurotoxicity in 3xTg Alzheimer's Mouse Model

doi: 10.1155/2019/1862437

Figure Lengend Snippet: Artemether increased phosphorylation of AMPK/GSK3 β (ser9)in PC12 cell cultures exposed to A β 1-42. (a, c) PC12 cells pretreated for 2 h with different concentrations of Artemether were incubated with 1 μ M A β 1-42 for 24 h .The lysates were submitted for measurements of phosphorylations by western blotting using specific antibodies for AMPK (P-AMPK), total AMPK (T-AMPK), GSK3 β (ser9) (P-GSK3 β ), and total GSK3 β (T-GSK3 β ). GAPDH was used for normalization. Three independent experiments were included. (b, d) Quantitation of phosphorylated kinases. ∗ p < 0.05 and ∗∗ p < 0.01 were considered significantly different.

Article Snippet: Anti-phospho-AMPK, anti-AMPK, anti-phospho-GSK3 β (ser9), anti-GSK3 β , anti-Nrf2, anti-HO-1, anti-phospho-tau, anti-tau, cleaved caspase 3, β -amyloid, anti-GFAP, and anti- β -actin antibodies were purchased from Cell Signaling Technology (Woburn, MA, USA).

Techniques: Incubation, Western Blot, Quantitation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemether Activation of AMPK/GSK3 β (ser9)/Nrf2 Signaling Confers Neuroprotection towards β- Amyloid-Induced Neurotoxicity in 3xTg Alzheimer's Mouse Model

doi: 10.1155/2019/1862437

Figure Lengend Snippet: Direct relationship between Artemether effects on AMPK/GSK3 β (ser9) phosphorylation and neuroprotection towards A β 1-42 -induced apoptotic cell death in PC12 cells. PC12 cells pretreated with 5 μ M compound C (AMPK inhibitor) for 30 min, followed by incubation with 1 μ M A β 1-42 in the presence or absence of different concentrations of Artemether. (a) Cell viability was measured by MTT assay. ∗ p < 0.05 and ∗∗∗ p < 0.001 were considered significantly different. (b) Apoptosis was determined by flow cytometry. (c) Quantitation of the percentage of cell apoptosis. ∗∗∗ p < 0.001 was considered significantly different. (d) Phosphorylation activities of AMPK/GSK3 β (ser9). GAPDH was used for normalization. (e, f) Quantitation of phosphorylation activities of AMPK/GSK3 β . All results are presented as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 were considered significantly different.

Article Snippet: Anti-phospho-AMPK, anti-AMPK, anti-phospho-GSK3 β (ser9), anti-GSK3 β , anti-Nrf2, anti-HO-1, anti-phospho-tau, anti-tau, cleaved caspase 3, β -amyloid, anti-GFAP, and anti- β -actin antibodies were purchased from Cell Signaling Technology (Woburn, MA, USA).

Techniques: Incubation, MTT Assay, Flow Cytometry, Quantitation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemether Activation of AMPK/GSK3 β (ser9)/Nrf2 Signaling Confers Neuroprotection towards β- Amyloid-Induced Neurotoxicity in 3xTg Alzheimer's Mouse Model

doi: 10.1155/2019/1862437

Figure Lengend Snippet: Artemether stimulated the phosphorylation of AMPK/GSK3 β (ser9) and increased the expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain cortex of 3xTg-AD mice. Artemether was administered to mice by intraperitoneal injection, once a day, at low doses of 5 mg/kg and high doses of 20 mg/kg for 4 weeks. Thereafter, the brain cortex extracts of wild-type (WT) compared to 3xTg-AD mice treated with either a low (Arte low) or high dose (Arte high) of Artemether or untreated (3xTg) were submitted for analyses. (a) Phosphorylations of AMPK/GSK3 β (ser9) and protein expression levels of Nrf2 and heme oxygenase-1 (HO-1); three mice per treatment group were used for western blot. (b–e) Quantitation of western blotting results of the different experimental groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 were considered significantly different.

Article Snippet: Anti-phospho-AMPK, anti-AMPK, anti-phospho-GSK3 β (ser9), anti-GSK3 β , anti-Nrf2, anti-HO-1, anti-phospho-tau, anti-tau, cleaved caspase 3, β -amyloid, anti-GFAP, and anti- β -actin antibodies were purchased from Cell Signaling Technology (Woburn, MA, USA).

Techniques: Expressing, Injection, Western Blot, Quantitation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemether Activation of AMPK/GSK3 β (ser9)/Nrf2 Signaling Confers Neuroprotection towards β- Amyloid-Induced Neurotoxicity in 3xTg Alzheimer's Mouse Model

doi: 10.1155/2019/1862437

Figure Lengend Snippet: A schematic diagram of Artemether-induced AMPK/GSK3 β (ser9)/Nrf2 activation towards reduction of amyloid- β -evoked oxidative stress to confer neuroprotection. Artemether stimulated AMPK/GSK3 β (ser9) phosphorylation output in neuronal cells and animal brain cortex to increase the nuclear expression of Nrf2 with induction of the phase II antioxidant enzymes and anti-inflammatory genes containing ARE. This process (dotted line) caused attenuation of APP-derived and A β -induced ROS production, reduction of oxidative stress, correction of mitochondrial dysfunction, reduction of inflammation, and conferring neuroprotection.

Article Snippet: Anti-phospho-AMPK, anti-AMPK, anti-phospho-GSK3 β (ser9), anti-GSK3 β , anti-Nrf2, anti-HO-1, anti-phospho-tau, anti-tau, cleaved caspase 3, β -amyloid, anti-GFAP, and anti- β -actin antibodies were purchased from Cell Signaling Technology (Woburn, MA, USA).

Techniques: Activation Assay, Expressing, Derivative Assay