Journal: International Journal of Molecular Sciences

Article Title: 5-Amino-1-β-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift

doi: 10.3390/ijms20133255

Figure Lengend Snippet: Effects of 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) on AMP-activated kinase (AMPK) activation, mechanical hyperalgesia, and paw edema in zymosan-induced paw inflammation. ( A ) The time course of mechanical hyperalgesia in wild type mice after injection of 10 mg/mL (20 µL) zymosan A into the hind paw with and without addition of AICAR (400 mg/kg i.p.). Zymosan was injected at time ‘0,’ and the response to mechanical stimulation was assessed hourly from 2 to 6 h with a dynamic plantar aesthesiometer. Delta paw withdrawal latency (dPWL) is the relative paw withdrawal latency expressed as a percentage difference between the treated (left) and untreated (right) hindpaws, calculated by the formula: (Left-right)/right × 100. ( n = 7, zymosan control; n = 8, zymosan + AICAR). Repeated measures ANOVA, * p = 0.0456, F(1,6) = 6.325. ( B ) Area under the paw volume versus time curve (AUC) from 2 to 6 h after zymosan A injection with and without treatment with AICAR (400 mg/kg body weight, i.p.), ( n ≥ 6/group). The paw volume was determined by plethysmometric analysis. *** p < 0.001 statistically significant difference in comparison with control. ( C ) The densitometric analysis of AMPKα1/2 phosphorylation in paws of mice with (grey column) and without AICAR treatment (black column) as assessed by western blot ( n ≥ 3/group), independent sample t -test,* p < 0.05 statistically significant difference in comparison with control. ( D ) The densitometric analysis of AMPKα1/2 phosphorylation in western blots with paws of control mice and ipsi- and contralateral paws of mice treated with zymosan ( n ≥ 3/group). Phosphorylated (p)-AMPK and AMPK protein levels were normalized with beta-actin, which served as loading control. Then, a ratio between pAMPK and AMPK was calculated.

Article Snippet: AICAR and metformin used as AMPK activators were purchased from Merck Chemicals Ltd. (Darmstadt, Germany).

Techniques: Activation Assay, Injection, Control, Comparison, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: 5-Amino-1-β-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift

doi: 10.3390/ijms20133255

Figure Lengend Snippet: Effects of AICAR and metformin on the phenotype of primary bone marrow derived macrophages. ( A ) FACS analysis showing a lipopolysaccharide (LPS)-induced shift towards a M1 phenotype indicated by an increase of CD86 positive cells. This phenotype switch is significantly inhibited by AICAR. ( n = 3). * p < 0.05, ( B ) Immunofluorescence showing CD86 and CD206 positive cells (both shown in red) in differentially treated bone marrow-derived macrophage (BMM) cultures. The cells were either incubated with vehicle, proinflammatory LPS, or antiinflammatory IL-4. Furthermore, the cells were co-incubated with LPS and AICAR, AICAR and adenosine kinase inhibitor ABT702, as well as metformin. IL-4-treated cells were also co-incubated with AICAR and metformin. The pictures show a representative analysis of three independent incubations. The cellular nuclei are visualized by DNA staining with 4′,6-Diamidin-2-phenylindol (DAPI) (blue). Scale Bar: 20 µm.

Article Snippet: AICAR and metformin used as AMPK activators were purchased from Merck Chemicals Ltd. (Darmstadt, Germany).

Techniques: Derivative Assay, Immunofluorescence, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: 5-Amino-1-β-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift

doi: 10.3390/ijms20133255

Figure Lengend Snippet: Effects of AICAR and metformin on the expression of inflammatory genes in primary bone marrow-derived macrophages. qRT-PCR analyses of proinflammatory IL-1beta, TNF alpha, iNOS, and antiinflammatory IL-10 and arginase 1 in LPS- and IL-4-stimulated BMMs with and without treatment with AICAR [500 µM] or metformin [500 µM], or AICAR in combination with its activation inhibitor ABT702, respectively. Signals for specific genes were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative quantitative level of samples was determined by standard 2 -dd C calculations and expressed as fold-change compared to a reference control sample. ( n = 3–4/treatment), * p < 0.05, ** p < 0.01, *** p < 0.001 statistically significant difference in comparison with LPS or IL-4-treated controls.

Article Snippet: AICAR and metformin used as AMPK activators were purchased from Merck Chemicals Ltd. (Darmstadt, Germany).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Activation Assay, Control, Comparison

Journal: bioRxiv

Article Title: AMPK-p38 axis converts human pluripotent stem cells to naïve state

doi: 10.1101/2022.03.31.486536

Figure Lengend Snippet: (A) Naïve conversion protocol using AICAR. PXGL; PD0325901 (1 μM), XAV939 (2 μM), Go6983 (2 μM), and human LIF (10 ng/ml) in Ndiff227 medium. (B) Appearance of EOS-GFP-positive cell clusters induced by AICAR (day14). (C) Flow-cytometry analysis of EOS-GFP, SUSD2, CD75, and CD57 expression (day14). (D) EOS-GFP-positive naïve-like colony after expansion in PXGL (day14+14p). (E) Flow-cytometry analysis of EOS-GFP, SUSD2 and CD75 expression (day14+21p). (F) RT-qPCR analysis of sorted SUSD2+CD75+ cells and parental primed hESCs (H1-EOS). AICAR: day14+13p and VPA: day9+4p. Error bars: S.D. of technical triplicates. (G) Immunostaining for OCT4, NANOG, KLF17, and TFE3 of primed and AICAR-induced cells. AICAR: day14+14p. (H) TMRE staining for mitochondrion dependent on mitochondrial membrane activity. Scale bars: 100 μm in B, D, H; 50 μm in G. AICAR: day14+16p.

Article Snippet: AICAR (Fujifilm, 1mM) was added to the PXGL medium (Liu et al., 2019; ), and VPA ( ) was added to Ndiff227 basal medium with PD0325901 (Sigma, 1 μM), human Lif (Fujifilm, 10 ng/ml), and penicillin/streptomycin) for naïve state induction.

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Immunostaining, Staining, Membrane, Activity Assay

Journal: bioRxiv

Article Title: AMPK-p38 axis converts human pluripotent stem cells to naïve state

doi: 10.1101/2022.03.31.486536

Figure Lengend Snippet: (A) Images of naïve hESCs (H1-EOS) induced by VPA. VPA, valproic acid (day9+6p). (B) Cell proliferation data of naive hESCs in the PXGL on MEF feeder cells. AICAR-naive 1: day14+19p, AICAR-naive 2: day14+20p, AICAR-naive 3: day14+7p at day0 in figure. (C) Images of naïve hiPSCs (Ff-I14-EOS) induced by AICAR(day14+7p) and Flow-cytometry analysis of EOS-GFP, SUSD2 and CD75 expression in naive hiPSCs (day14+7p). Scale bars: 100 μm

Article Snippet: AICAR (Fujifilm, 1mM) was added to the PXGL medium (Liu et al., 2019; ), and VPA ( ) was added to Ndiff227 basal medium with PD0325901 (Sigma, 1 μM), human Lif (Fujifilm, 10 ng/ml), and penicillin/streptomycin) for naïve state induction.

Techniques: Flow Cytometry, Expressing