Journal: Diabetes & Metabolism Journal

Article Title: Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis

doi: 10.4093/dmj.2022.0110

Figure Lengend Snippet: EW-7197 relieves hepatocellular fibrosis but has side effects. Hepatocellular fibrosis was induced by treating human hepatic stellate cell (LX-2) cells with transforming growth factor β (TGF-β; 2 ng/mL). (A, B) The ability of EW-7197 to restore the original state was tested by expressing fibrosis markers such as p-SMAD2/3, α-smooth muscle actin (α-SMA), and ColI. (C, D) Alpha mouse liver 12 (AML12) cells were treated with EW-7197 combined with TGF-β. (E, F) AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (Erk), and Akt activities were measured to determine the effect of EW-7197 treatment on the TGF-β non-canonical pathway. a P <0.05 vs. control (Con), b P <0.05 vs. TGF-β, c P <0.001 vs. Con, d P <0.001 vs. TGF-β.

Article Snippet: Antibodies against p-SMAD2 (#3108), p-SMAD3 (#9520), phosphor-AMPK (p-AMPK) (#2531), AMPK (#2532), and Kelch-like ECH-associated protein 1 (Keap1) (#4678) were obtained from Cell Signaling Technologies.

Techniques: Expressing

Journal: Diabetes & Metabolism Journal

Article Title: Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis

doi: 10.4093/dmj.2022.0110

Figure Lengend Snippet: Curcumin 2005-8 (Cur5-8) and EW-7197 co-administration improves hepatic lipid accumulation. (A, B) Accumulation of lipid droplets and variations in liver sizes were measured using H&E staining and liver imaging, respectively. (C) Nonalcoholic fatty liver disease (NAFLD) activity was scored by quantifying the degree of steatosis, hepatocyte ballooning, and lobular inflammation based on the results of H&E staining. (D, E, F) The NAFLD activity score is assigned as described in the Methods section. Metabolism-related proteins were determined by measuring rho-associated coiled-coil kinase 1 (Rock1) and sterol regulatory elementbinding protein 1c (Srebp1c) levels, AMP-activated protein kinase (AMPK) activity, and reactive oxygen species (ROS) scavenging effect. Endoplasmic reticulum stress regulation was determined by measuring Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and activating transcription factor 6α (ATF6α) levels. a P <0.05 vs. control (Con), b P <0.05 vs. methionine-choline-deficient diet (MCD), c P <0.001 vs. Con, d P <0.001 vs. MCD.

Article Snippet: Antibodies against p-SMAD2 (#3108), p-SMAD3 (#9520), phosphor-AMPK (p-AMPK) (#2531), AMPK (#2532), and Kelch-like ECH-associated protein 1 (Keap1) (#4678) were obtained from Cell Signaling Technologies.

Techniques: Staining, Imaging, Activity Assay

Journal: Veterinary Research

Article Title: Curcumin-dependent phenotypic transformation of microglia mediates resistance to pseudorabies-induced encephalitis

doi: 10.1186/s13567-023-01149-x

Figure Lengend Snippet: CUR regulates energy metabolism through AMPK-dependent pathways in PRV-infected BV2 cells. A BV2 cells were infected with/without PRV for 24 h and then treated with/without 20 μM CUR for 24 h. Western blot analysis of p-AMPK Thr172 protein levels (normalized to the t-AMPK protein). β-Actin was used as the loading control ( n = 3). B Relative protein levels of p-AMPK Thr172 . C BV2 cells were treated with different concentrations of Compound C for 24 h, and cell viability was determined by the CCK-8 assay to determine the toxicity range ( n = 4). D BV2 cells were infected with/without PRV or were treated with PRV and an AMPK inhibitor (Compound C or siRNA) alone or in combination for 24 h, followed by treatment with/without 20 μM CUR for 24 h. Western blot analysis of p-AMPK (normalized to the t-AMPK protein), t-AMPK, Gpat4, and LDHa levels (Gpat4 and LDHa values were normalized to the β-actin protein); β-actin was used as the loading control ( n = 3). E Relative p-AMPKThr172 protein levels. F Relative LDHa protein levels. G Relative Gpat4 protein levels. H The extracellular acidification rate of BV2 cells after the different treatments (values normalized to the control) ( n = 4). I The OCR of BV2 cells after the different treatments (values normalized to the control) ( n = 4). J BV2 cell ATP levels in the different treatment groups ( n = 4). K The M1 phenotype-related inflammatory factor TNF-α in BV2 cells was detected using ELISA ( n = 4). L The levels of the M1 phenotype-related inflammatory factor IL-6 in BV2 cells ( n = 4). M The levels of the M2 phenotype-related anti-inflammatory factor IL-4 in BV2 cells ( n = 4). N The levels of the M2 phenotype-related anti-inflammatory factor IL-10 in BV2 cells ( n = 4). All experiments were performed in parallel. The results are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA followed by an LSD post hoc test for multiple comparisons among the groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, not significant.

Article Snippet: Furthermore, the membrane was blocked with 5% nonfat dry milk or 5% BSA in tris-buffered saline containing Tween-20 (TBST) at 25 ℃ and then incubated overnight at 4 °C with the following antibodies: rabbit anti-AMPK (1:2000, bs-1115R, Bioss, China), phosphor(p)-AMPK Thr172 (1:1000, 2531, Cell Signaling, USA), lactate dehydrogenase A (LDHa, 1:1000, WL03271, Wanleibio, China), nuclear factor kappa-B p65 (NF-κB p65, 1:1000, WL01273b, Wanleibio, China), p-NF-κB p65 Ser536 (1:10 000, WL02169, Wanleibio, China), glycerol-3-phosphate acyltransferase 4 (GPAT4, 1:1000, bs-15587R, Bioss, China), Bax (1:10 000, 50,599–2-Ig, Proteintech, China), Caspase-3/p17/p19 (1:1000, 19,677–1-AP, Proteintech, China), Bcl-2 (1:4000, 26,593–1-AP, Proteintech, China), and β-actin (1:5000, 20,536–1-AP, Proteintech, China).

Techniques: Infection, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0093256

Figure Lengend Snippet: PLC/PRF/5 cancer cells (2×10 6 ) were inoculated s.c. in 5-week old male nu/nu mice at both flanks for 4 points. After 1 week, metformin was dissolved in PBS and given i.p. (30 Ag/g body weight). The control group received PBS only. Control and metformin-treated groups were treated for 7 weeks. (A): Representative picture for tumorigenesis number and size in control and metformin treatment nu/nu mice. (B): The tumorigenesis percentage. (C): Average tumor weight. (D): Western blot analysis of phosphor-AMPK (AMPK-p) and AMPK in control and metformin treated groups. (E): The expression of Ki-67 in control and metformin-treated groups by immunohistochemistry. Data are mean±SD. *p<0.05.

Article Snippet: The membranes were immunoblotted with anti-phosphor-AMPK antibody (AMPK-p) (Cell signaling Technology, CA, United States), anti-AMPK antibody (Cell signaling Technology, CA, United States), anti-P21 CIP antibody (Maixin Bio, Fuzhou, China), anti-p27 KIP antibody (Maixin Bio, Fuzhou, China) or anti-cyclin D1 antibody (Maixin Bio, Fuzhou, China), followed by the secondary antibody horseradish peroxidase-conjugated IgG (Vector Laboratories, Burlingame, CA, United States).

Techniques: Western Blot, Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0093256

Figure Lengend Snippet: (A) Positive signals were detected in cytoplasm and occasionally in nucleus. (B) Negative control (omitting primary antibody). (C)Representative images of three samples of HCC and paracancerous liver tissue showing AMPK activity. (D) Relative AMPK activity in 30 tissue samples. Data are mean±SD. *p<0.05 (E) Western blot analysis of phosphor-AMPK (AMPK-p) and AMPK. Representative bands from five independent experiments.

Article Snippet: The membranes were immunoblotted with anti-phosphor-AMPK antibody (AMPK-p) (Cell signaling Technology, CA, United States), anti-AMPK antibody (Cell signaling Technology, CA, United States), anti-P21 CIP antibody (Maixin Bio, Fuzhou, China), anti-p27 KIP antibody (Maixin Bio, Fuzhou, China) or anti-cyclin D1 antibody (Maixin Bio, Fuzhou, China), followed by the secondary antibody horseradish peroxidase-conjugated IgG (Vector Laboratories, Burlingame, CA, United States).

Techniques: Negative Control, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0093256

Figure Lengend Snippet: The correlation between AMPK-p and hemorrhage and/or necrosis in HCC.

Article Snippet: The membranes were immunoblotted with anti-phosphor-AMPK antibody (AMPK-p) (Cell signaling Technology, CA, United States), anti-AMPK antibody (Cell signaling Technology, CA, United States), anti-P21 CIP antibody (Maixin Bio, Fuzhou, China), anti-p27 KIP antibody (Maixin Bio, Fuzhou, China) or anti-cyclin D1 antibody (Maixin Bio, Fuzhou, China), followed by the secondary antibody horseradish peroxidase-conjugated IgG (Vector Laboratories, Burlingame, CA, United States).

Techniques:

Journal: PLoS ONE

Article Title: AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0093256

Figure Lengend Snippet: The correlation between AMPK-p and the clinical features of HCC patients.

Article Snippet: The membranes were immunoblotted with anti-phosphor-AMPK antibody (AMPK-p) (Cell signaling Technology, CA, United States), anti-AMPK antibody (Cell signaling Technology, CA, United States), anti-P21 CIP antibody (Maixin Bio, Fuzhou, China), anti-p27 KIP antibody (Maixin Bio, Fuzhou, China) or anti-cyclin D1 antibody (Maixin Bio, Fuzhou, China), followed by the secondary antibody horseradish peroxidase-conjugated IgG (Vector Laboratories, Burlingame, CA, United States).

Techniques: