p ampk α 1 2  (Santa Cruz Biotechnology)

Journal: Chinese Herbal Medicines

Article Title: Ganoderic acid a derivative induces apoptosis of cervical cancer cells by inhibiting JNK pathway

doi: 10.1016/j.chmed.2024.07.002

Figure Lengend Snippet: (A) Target prediction analysis results. (B) Prediction of GaAD19-JNK interaction. (C–E) Western blot analysis of HeLa cells with GaAD19 treatment for 24 h on expression of JNK, p-JNK, ERK, p-ERK, p38, p-p38 (C), AMPK α 1, p-AMPK α 1-S485, p-AMPK α 1-S496, AMPK β 1, p-AMPK β 1-S108, Akt, p-Akt (D), MAP2K4, MAP2K7 (E), and other proteins (means ± SD, n = 3). (F) Effect of GaAD19 on mRNA level of JNK pathway membrane protein receptor in HeLa cells was determined by qPCR (means ± SD, n = 3). ( ** P < 0.01, *** P < 0.001 vs control group, ns indicates no significant difference).

Article Snippet: AMPK α 1 , Proteintech , 10929–2-AP , 1:200.

Techniques: Western Blot, Expressing, Membrane, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: IMM-H007 promotes hepatic cholesterol and triglyceride metabolism by activating AMPK α to attenuate hypercholesterolemia

doi: 10.1016/j.apsb.2025.05.015

Figure Lengend Snippet: The increase in LDLR protein levels induced by IMM-H007 depends on AMPK α 1/2. (A) LDLR protein levels in Apoe −/− mouse livers were determined via immunofluorescence staining, and the mean immunofluorescence intensity (MFI) of the images was quantified. Scale bar = 50 μm. n = 5. (B) HepG2 cells were treated with H007-M1 at the indicated concentrations for 24 h. LDLR protein expression was determined by Western blotting. (C) HepG2 cells were pretreated with H007-M1 (100 μmol/L) for 12 h and then incubated with CHX (100 μmol/L) for 0, 3, or 6 h. LDLR protein expression was determined by Western blot (top), and the half-life was calculated (bottom). (D, E) HepG2 cells were transfected with scrambled siRNA or AMP-activated protein kinase ( Ampk ) a 1 siRNA and treated with H007-M1 (100 μmol/L) for 24 h. LDLR and AMPK α 1 protein expression was subsequently determined via Western blotting (D), and the band density (E) was quantified. (F, G) HepG2 cells were transfected with scrambled siRNA or AMPKA 2 siRNA and treated with H007-M1 (100 μmol/L) for 24 h, after which LDLR and AMPK α 2 protein expression was determined by Western blotting (F), and the band density was quantified (G). (H, I) HepG2 cells were transfected with scrambled siRNA or AMPKG siRNA and treated with H007-M1 (100 μmol/L) for 24 h, after which LDLR and AMPK γ protein expression was determined by Western blotting (H), and the band density was quantified (I). (J, K) HepG2 cells were transfected with scrambled siRNA or AMPKB siRNA and treated with H007-M1 (100 μmol/L) for 24 h. LDLR and AMPK β protein expression levels were subsequently determined via Western blotting (J), and the band density (K) was quantified. Statistical data are presented as mean ± SEM. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus the indicated group ( n = 3).

Article Snippet: Rabbit anti-LDLR (Cat# 10785-1-AP), PCSK9 (Cat# 55206-1-AP), ABCG1 (Cat# 13578-1-AP), AMPK α 2 (Cat# 18167-1-AP), AMPK β (Cat# 10308-1-AP), AMPK γ (Cat# 10290-1-AP), FXR (Cat# 25055-1-AP), CREB1 (Cat# 12208-1-AP) polyclonal antibodies, mouse anti-FOXO1 (Cat# 66457-1), and HRP-conjugated GAPDH monoclonal antibodies (Cat# HRP-60004), TRITC–conjugated Goat Anti-Rabbit IgG (Cat# SA00007-2) were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Incubation, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: IMM-H007 promotes hepatic cholesterol and triglyceride metabolism by activating AMPK α to attenuate hypercholesterolemia

doi: 10.1016/j.apsb.2025.05.015

Figure Lengend Snippet: Hepatocyte Ampka 1/2 deficiency blocked the effect of IMM-H007 on hypercholesterolemia. (A) Ampka flox/flox and h Ampka KO mice were fed a high-fat (HF)/high-cholesterol (HC)/bile-salt (BS) diet with or without IMM-H007 (200 mg/kg body weight/day) for 5 weeks. (B) Determination of serum TC. (C) Determination of serum non-HDL-C. (D) Determination of serum HDL-C levels. (E) Determination of serum TG levels. Statistical data are presented as mean ± SEM. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus the indicated group ( n = 5). (F) RNA-seq assay with mouse liver total RNA. Enrichment analysis of the KEGG and REACTOME pathways ( n = 3). (G, H) Protein levels of LDLR (G, scale bar = 100 μm) AMPK α 1/2 (H, scale bar = 200 μm) in mouse liver was determined by immunofluorescent staining.

Article Snippet: Rabbit anti-LDLR (Cat# 10785-1-AP), PCSK9 (Cat# 55206-1-AP), ABCG1 (Cat# 13578-1-AP), AMPK α 2 (Cat# 18167-1-AP), AMPK β (Cat# 10308-1-AP), AMPK γ (Cat# 10290-1-AP), FXR (Cat# 25055-1-AP), CREB1 (Cat# 12208-1-AP) polyclonal antibodies, mouse anti-FOXO1 (Cat# 66457-1), and HRP-conjugated GAPDH monoclonal antibodies (Cat# HRP-60004), TRITC–conjugated Goat Anti-Rabbit IgG (Cat# SA00007-2) were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: RNA Sequencing, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: IMM-H007 promotes hepatic cholesterol and triglyceride metabolism by activating AMPK α to attenuate hypercholesterolemia

doi: 10.1016/j.apsb.2025.05.015

Figure Lengend Snippet: IMM-H007 upregulates LDLR expression via the AMPK α 1/2–SENP1–IDOL signaling pathway. (A, B) HepG2 cells were posttranslationally transfected with scrambled siRNA, proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) siRNA (A), or inducible degrader of LDLR ( IDOL ) siRNA (B), after which LDLR, PCSK9, and IDOL protein expression was determined by Western blotting. (C) mRNA levels of PCSK9 and IDOL in HepG2 cells after H007-M1 treatment were measured via qRT-PCR ( n = 3). (D) HEK-293T cells were transfected with Flag- IDOL , HA-small ubiquitin-like modifier 1 ( SUMO1 ), or His-ubiquitin-conjugating enzyme ( UBC9 ) for 24 h and then treated with H007-M1 (100 μmol/L) for 24 h. The SUMOylation of Flag- IDOL was determined by an IP assay using Flag beads. (E) HepG2 cells were transfected with scrambled siRNA or SUMO-specific protease 1 ( SENP1 ) siRNA, after which the protein expression of IDOL, SENP1, and LDLR was determined by Western blotting. (F) Cas9-Ctrl and Cas9- AMPKA cells were treated with H007-M1 at the indicated concentrations for 24 h. IDOL, SENP1, and AMPKA protein expression was determined by Western blotting. (G) Protein expression in the livers of the mice in A was determined by Western blotting. (H) mRNA levels of SENP1 in HepG2 cells after H007-M1 treatment or transfection with AMPKA 1/2 siRNA were measured via qRT-PCR ( n = 3). (I) Putative cAMP response element ( CRE) region in the h SENP1 promoter and the mutated sites. (J) HepG2 cells were transfected with the SENP1 promoter or SENP1 - CRE -mut promoter and Renilla (as an internal control) overnight, followed by 100 μmol/L H007-M1 treatment or AMPKA 1/2 overexpression for another 24 h. The activity of firefly and Renilla luciferases in the cellular lysate was determined via a dual-luciferase reporter assay ( n = 8). Statistical data are presented as mean ± SEM. ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001; NS, not significant versus the indicated groups.

Article Snippet: Rabbit anti-LDLR (Cat# 10785-1-AP), PCSK9 (Cat# 55206-1-AP), ABCG1 (Cat# 13578-1-AP), AMPK α 2 (Cat# 18167-1-AP), AMPK β (Cat# 10308-1-AP), AMPK γ (Cat# 10290-1-AP), FXR (Cat# 25055-1-AP), CREB1 (Cat# 12208-1-AP) polyclonal antibodies, mouse anti-FOXO1 (Cat# 66457-1), and HRP-conjugated GAPDH monoclonal antibodies (Cat# HRP-60004), TRITC–conjugated Goat Anti-Rabbit IgG (Cat# SA00007-2) were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Control, Over Expression, Activity Assay, Luciferase, Reporter Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: IMM-H007 promotes hepatic cholesterol and triglyceride metabolism by activating AMPK α to attenuate hypercholesterolemia

doi: 10.1016/j.apsb.2025.05.015

Figure Lengend Snippet: IMM-H007 promotes hepatic cholesterol metabolism by activating the AMPK α 1–FXR pathway. (A) Heatmap of the differentially expressed genes related to bile acid metabolism in the liver identified via RNA-seq. (B) Cholesterol metabolism-related protein expression in the livers of the mice shown in A was determined by Western blotting. (C) 293T cells were transfected with the farnesoid X receptor ( FXR ) promoter and Renilla (as an internal control) overnight, followed by 100 μmol/L H007-M1 treatment for another 24 h. The activity of firefly and Renilla luciferases in the cellular lysate was determined via a dual-luciferase reporter assay ( n = 5). (D, E) HepG2 cells were transfected with scrambled siRNA or AMPKA1 siRNA and treated with H007-M1 (100 μmol/L) for 24 h. Then, protein expression was determined by Western blotting (D), and the band density (E) was quantified. (F, G) HepG2 cells were transfected with scrambled siRNA or AMPKA2 siRNA and treated with H007-M1 (100 μmol/L) for 24 h. Then, protein expression was determined by Western blotting (F), and the band density was quantified (G) ( n = 3). (H) Hepatic cholesterol quantitative analysis of the mice in A. (I) Hepatic total bile acid (TBA) quantitative analysis of the mice in A ( n = 5). Statistical data are presented as mean ± SEM. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; NS, not significant versus the indicated group.

Article Snippet: Rabbit anti-LDLR (Cat# 10785-1-AP), PCSK9 (Cat# 55206-1-AP), ABCG1 (Cat# 13578-1-AP), AMPK α 2 (Cat# 18167-1-AP), AMPK β (Cat# 10308-1-AP), AMPK γ (Cat# 10290-1-AP), FXR (Cat# 25055-1-AP), CREB1 (Cat# 12208-1-AP) polyclonal antibodies, mouse anti-FOXO1 (Cat# 66457-1), and HRP-conjugated GAPDH monoclonal antibodies (Cat# HRP-60004), TRITC–conjugated Goat Anti-Rabbit IgG (Cat# SA00007-2) were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: RNA Sequencing, Expressing, Western Blot, Transfection, Control, Activity Assay, Luciferase, Reporter Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: IMM-H007 promotes hepatic cholesterol and triglyceride metabolism by activating AMPK α to attenuate hypercholesterolemia

doi: 10.1016/j.apsb.2025.05.015

Figure Lengend Snippet: Hepatocyte AMPKα 1/2 deficiency blocked the effect of IMM-H007 on lipid accumulation in the liver. (A) Liver photos of the mice in A. (B, C) H&E (B) and ORO (C) staining of liver sections from the mice in A. Scale bar = 100 μm. (D) TG quantitative analysis of total liver lipid extracts from the mice in A ( n = 5). (E) Adipose triglyceride lipase ( ATGL ) mRNA expression in the livers of the mice shown in A was determined by qRT-PCR ( n = 5). (F) ATGL, diacylglycerol O -acyltransferase 1 (DGAT1) protein expression in the livers of the mice in A was determined by Western blotting ( n = 3). (G) Cas9-Ctrl and Cas9- AMPKA HepG2 cells were treated with H007-M1 at the indicated concentrations for 12 h. Protein expression was determined by Western blotting ( n = 3). (H) Schematic diagram of the potential mechanism by which IMM-H007 decreases hepatic lipid accumulation. Statistical data are presented as mean ± SEM. ∗ P < 0.05 versus the indicated groups.

Article Snippet: Rabbit anti-LDLR (Cat# 10785-1-AP), PCSK9 (Cat# 55206-1-AP), ABCG1 (Cat# 13578-1-AP), AMPK α 2 (Cat# 18167-1-AP), AMPK β (Cat# 10308-1-AP), AMPK γ (Cat# 10290-1-AP), FXR (Cat# 25055-1-AP), CREB1 (Cat# 12208-1-AP) polyclonal antibodies, mouse anti-FOXO1 (Cat# 66457-1), and HRP-conjugated GAPDH monoclonal antibodies (Cat# HRP-60004), TRITC–conjugated Goat Anti-Rabbit IgG (Cat# SA00007-2) were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I restore intestinal barrier function by targeting the S100A9/AMPK/mTOR signaling pathway

doi: 10.3389/fphar.2025.1530109

Figure Lengend Snippet: S100A9 regulated the expression of TJ proteins in colonic epithelial cells through AMPK/mTOR pathway. (A,B) NCM460 cells were pretreated with AT-1 (10 μM) for 1 h, followed with S100A9 (1 μg/mL) for 24 h, then detected by flow cytometry assay. (C–F) NCM460 cells were pretreated with AT-1 (10 μM) for 1 h, followed with S100A9 (1 μg/mL) for 24 h, the level of ZO-1, claudin-1, p38MAPK, p-p38MAPK, JNK,p-JNK, ERK1/2, p-ERK1/2, AMPKα, p-AMPKα, mTOR, p-mTOR, and MLCK, was evaluated by Western blot. ### p < 0.001 vs. Blank group; * p < 0.05 and *** p < 0.001 vs . S100A9 group.

Article Snippet: Atractylenolide-I (AT-1, C15H18O2, MW:230.32, ≥98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); sodium butyrate (NaB, C4H7NaO2, MW:110.09, ≥98% purity) was purchased from Macklin Biochemical Technology (Shanghai, China); dextran sulfate sodium (DSS) was purchased from MP Biomedicals (Solon, OH, USA); myeloperoxidase (MPO) Kit was purchased from Jiangsu Meimian industrial Co., Ltd (Nanjing, China); fluorescein isothiocyanate-labeled dextran (FD4, average mol wt 3,000–5,000) was purchased from Sigma Aldrich (St. Louis, MO, USA); recombinant human S100A9 protein, dorsomorphin and BAPTA-AM were purchased from Med Chem Express (Shanghai, China); ZO-1, occludin, claudin-1, MLCK, β-actin, HA, mTOR, AMPK-α, ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), Flag primary antibodies, HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were purchased from Proteintech (Wuhan, China); SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), phospho-AMPKα (Thr172), myosin light chain 2, phospho-myosin light chain 2 (Thr18/Ser19) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); rhodamine (TRITC) Affinipure goat anti-mouse IgG(H+L), FITC-AffiniPure rabbit anti-rabbit IgG (H+L) were purchased from Yeasen Biotechnology (Shanghai, China); DMEM, RPMI 1640 medium, pen/strep, fetal bovine serum (FBS), opti-MEM medium were purchased from Gibco BRL (Gaithersburg, MD, USA); total protein extraction kit were purchased from KeyGenbio (Nanjing, China); fastPure complex tissue/cell total RNA isolation kit, hiScriptTM QRTSuperMix and AceQTMqPCR SYBR green master mix were purchased from Vazyme Biotech (Nanjing, China); lipofectamineTM 3000 transfection reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA); quickBlockTM blocking buffer for immunol staining was purchased from Beyotime (Shanghai, China); PBS, rapid sealing solution (Protein free) were purchased from Servicebio (Wuhan, China); other chemical products used were of the analytical grade available.

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I restore intestinal barrier function by targeting the S100A9/AMPK/mTOR signaling pathway

doi: 10.3389/fphar.2025.1530109

Figure Lengend Snippet: AT-1 regulated the expression of TJ proteins in colonic epithelial cells through AMPK/mTOR pathway. (A,B) NCM460 cells were pretreated with Dorsomorphin(200 ng/mL) and AT-1 (10 μM) for 1 h, followed with S100A9 (1 μg/mL) for 24 h, the level of ZO-1, claudin-1, AMPKα, p- AMPKα, mTOR, p-mTOR, was evaluated by Western blot. (C) NCM460 cells were pretreated with BAPTA-AM (5 μM) and AT-1 (10 μM) for 1 h, followed with S100A9 (1 μg/mL) for 24 h, then detected by flow cytometry assay. (D,E) NCM460 cells were pretreated with BAPTA-AM (5 μM) and AT-1 (10 μM) for 1 h, followed with S100A9 (1 μg/mL) for 24 h, the level of ZO-1, claudin-1, AMPKα, p- AMPKα, mTOR, p-mTOR, was evaluated by Western blot. ###P < 0.001 vs. Blank group; * p < 0.05 and *** p < 0.001 vs. S100A9 group.

Article Snippet: Atractylenolide-I (AT-1, C15H18O2, MW:230.32, ≥98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); sodium butyrate (NaB, C4H7NaO2, MW:110.09, ≥98% purity) was purchased from Macklin Biochemical Technology (Shanghai, China); dextran sulfate sodium (DSS) was purchased from MP Biomedicals (Solon, OH, USA); myeloperoxidase (MPO) Kit was purchased from Jiangsu Meimian industrial Co., Ltd (Nanjing, China); fluorescein isothiocyanate-labeled dextran (FD4, average mol wt 3,000–5,000) was purchased from Sigma Aldrich (St. Louis, MO, USA); recombinant human S100A9 protein, dorsomorphin and BAPTA-AM were purchased from Med Chem Express (Shanghai, China); ZO-1, occludin, claudin-1, MLCK, β-actin, HA, mTOR, AMPK-α, ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), Flag primary antibodies, HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were purchased from Proteintech (Wuhan, China); SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), phospho-AMPKα (Thr172), myosin light chain 2, phospho-myosin light chain 2 (Thr18/Ser19) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); rhodamine (TRITC) Affinipure goat anti-mouse IgG(H+L), FITC-AffiniPure rabbit anti-rabbit IgG (H+L) were purchased from Yeasen Biotechnology (Shanghai, China); DMEM, RPMI 1640 medium, pen/strep, fetal bovine serum (FBS), opti-MEM medium were purchased from Gibco BRL (Gaithersburg, MD, USA); total protein extraction kit were purchased from KeyGenbio (Nanjing, China); fastPure complex tissue/cell total RNA isolation kit, hiScriptTM QRTSuperMix and AceQTMqPCR SYBR green master mix were purchased from Vazyme Biotech (Nanjing, China); lipofectamineTM 3000 transfection reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA); quickBlockTM blocking buffer for immunol staining was purchased from Beyotime (Shanghai, China); PBS, rapid sealing solution (Protein free) were purchased from Servicebio (Wuhan, China); other chemical products used were of the analytical grade available.

Techniques: Expressing, Western Blot, Flow Cytometry