Journal: The Journal of Biological Chemistry

Article Title: Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site

doi: 10.1016/j.jbc.2022.101852

Figure Lengend Snippet: Effects of Lusianthridin in intact cells. A , U2OS cells were treated with Lusianthridin, AICAR, or A-769662 for 30 min before lysing and performing an HTRF assay with the phospho-ACC (pACC) assay kit, and results are presented as the ratio of 665/620 nm (pACC HTRF). B , Western blot analysis of U2OS cells treated with Lusianthridin or AICAR with the indicated antibodies. Representative blots of n = 3 are shown along with the quantification of the pACC and pThr172 antibodies. C , U2OS cells were treated with varying concentrations of Lusianthridin for 30 min with or without prior treatment with STO-609 (10 μg/ml, 1 h). pACC was determined by performing an HTRF assay with the pACC assay kit, and results are presented as the ratio of 665/620 nm (pACC HTRF). A representative blot is displayed for the ionomycin-treated cell lysates using an automated capillary immunoblotting system (Sally Sue). AICAR, 5-aminoimidazole-4-carboxamide riboside; HTRF, Homogenous Time-Resolved Fluorescence.

Article Snippet: HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Techniques: HTRF Assay, Western Blot, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site

doi: 10.1016/j.jbc.2022.101852

Figure Lengend Snippet: Effects of Lusianthridin in β1 KO U2OS cells. pACC HTRF analysis of WT and β1 KO cells treated with AICAR ( A ), A-769662 ( B ), or Lusianthridin ( C ). Results are presented as the fold increase in pACC HTRF signal, means ± SEM of three independent experiments. D , Western blot (WB) analysis of WT and β1 KO cells treated with Lusianthridin using the indicated antibodies. Quantification of three independent experiments is shown as a ratio of the signal of pACC/ACC, along with a representative blot. AICAR, 5-aminoimidazole-4-carboxamide riboside; HTRF, Homogenous Time-Resolved Fluorescence; pACC, phospho-ACC.

Article Snippet: HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Techniques: Western Blot, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site

doi: 10.1016/j.jbc.2022.101852

Figure Lengend Snippet: Effects of Lusianthridin in β1/2 double KO (DKO) U2OS cells stably expressing β1 or β2 WT and β1 or β2 S108A. A , Western blot analysis of β1/2 DKO cells stably expressing β1 or β2 WT and β1 or β2 S108A using the indicated antibodies. B – D , pACC HTRF analysis of stable cell lines expressing β1 or β2 WT and β1 or β2 S108A treated with AICAR ( B ), A-769662 ( C ), or Lusianthridin ( D ). Results are presented as the fold increase in pACC HTRF signal and are means ± SEM of at least three independent experiments. E , Western blot analysis of β1 WT and β1 S108A stable cell lines treated with Lusianthridin using the indicated antibodies. Quantification of three independent experiments is shown as a ratio of the signal of pACC/ACC, along with a representative blot. AICAR, 5-aminoimidazole-4-carboxamide riboside; HTRF, Homogenous Time-Resolved Fluorescence; pACC, phospho-ACC.

Article Snippet: HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Techniques: Stable Transfection, Expressing, Western Blot, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site

doi: 10.1016/j.jbc.2022.101852

Figure Lengend Snippet: Lusianthridin activates pACC and inhibits de novo lipogenesis in primary mouse hepatocytes . Dose-dependent inhibition of de novo lipogenesis by AICAR ( A ) or Lusianthridin ( B ) after treatment of hepatocytes for 1 h. Western blot analysis of hepatocytes treated with AICAR or Lusianthridin is shown under the graph. Quantification of pACC/ACC ratio is displayed on the left Y -axis along with the percentage lipogenesis on the right Y -axis as the mean ± SEM from at least three independent experiments. C , inhibition of de novo lipogenesis in mouse primary hepatocytes isolated from WT or ACC1 (S79A)/2 (S212A) double KI mice (ACC DKI). Results are displayed as a percentage of lipogenesis and are the mean ± SEM from at least two independent experiments. D and E , Western blot analysis of mouse primary hepatocytes isolated from WT or S108A KI (S108A) mice after treatment with the indicated concentrations of AICAR ( D ) or Lusianthridin ( E ). Phosphorylation of ACC was quantified and normalized to total ACC protein (pACC/ACC). The fold increase (mean ± SEM)) in pACC/ACC is displayed above a representative blot of at least three independent experiments. F , inhibition of lipogenesis was determined in WT or S108A KI hepatocytes treated with the indicated concentrations of Lusianthridin or AICAR. Results are displayed as a percentage of lipogenesis and are the mean ± SEM from at least three independent experiments. AICAR, 5-aminoimidazole-4-carboxamide riboside; pACC, phospho-ACC.

Article Snippet: HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Techniques: Inhibition, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite–binding site

doi: 10.1016/j.jbc.2022.101852

Figure Lengend Snippet: The phenanthrene, Lusianthrin, also directly activates AMPK through the ADaM-binding site . A , structure of Lusianthrin. B , AMPK activity of bacterially expressed recombinant α2β1γ1 in the presence of increasing concentrations of Lusianthridin or Lusianthridin. AMPK activity was determined using the HTRF KinEASE assay kit, and results are presented as fold increase in AMPK activity (±SEM, n = 3) relative to the activity in the absence of activator. C and D , pACC HTRF analysis of stable cell lines expressing β1 WT/S108A ( C ) or β2 WT/S108A ( D ) treated with Lusianthrin. Results are presented as the fold increase in pACC HTRF signal and are means ± SEM of at least three independent experiments. ADaM, allosteric drug and metabolite; AMPK, AMP-activated protein kinase; HTRF, Homogenous Time-Resolved Fluorescence; pACC, phospho-ACC.

Article Snippet: HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Techniques: Binding Assay, Activity Assay, Recombinant, Stable Transfection, Expressing, Fluorescence