Journal: ACS Pharmacology & Translational Science

Article Title: Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin

doi: 10.1021/acsptsci.8b00002

Figure Lengend Snippet: (a) Receptor subunit composition, (b) activation of signaling pathways at the corresponding receptors by human amylin (hAMY), (c) activation of signaling pathways at the corresponding receptors by all peptides. In panel b, the concentration response curves are the combined mean data from four or five independent experiments (cAMP, pCREB n = 5, IP1, pERK1/2 n = 4). In panel c, potency data are summarized in radial plots showing mean pEC50 values from between three and five individual experiments. Exact experimental n is provided in Tables SB1–4. All errors are s.e.m. pERK1/2 data is the 15 min time point.

Article Snippet: Phosphorylated (p) extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB were detected using the AlphaLISA SureFire Ultra pERK1/2 (Thr202/Tyr204) or the AlphaLISA SureFire Ultra pCREB (Ser133) assay kits (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) as per the manufacturer’s protocol.

Techniques: Activation Assay, Concentration Assay

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 3. 6-OHDA induced autophagy is associated with modulation of AMPK/mTOR signaling. (A) SH-SY5Y cells were incubated with 6-OHDA (50 μM) and the activation of AMPK/ mTOR signaling molecules was assessed by immunoblotting at the indicated time points. The blots from a representative of three experiments are presented with the densitometry data above the relevant bands. (B) Control shRNA- or AMPK shRNA-transfected cells were treated with 6-OHDA for 16 h and the intracellular acidification was analyzed by flow cytometry after acridine orange (AO) staining (the insert shows immunoblot confirmation of AMPK knockdown). The data are mean±SD values from three independent experiments (#pb0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control). (C) Control shRNA- or AMPK shRNA-transfected cells were treated with 6-OHDA (50 μM) for 8 h and the activation of AMPK/mTOR signaling molecules, LC3 conversion and p62 levels were assessed by immunoblotting. The blots from one of at least two experiments with similar results are presented with the densitometry data above the relevant bands.

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Activation Assay, Western Blot, Control, shRNA, Transfection, Cytometry, Staining, Knockdown

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 4. Autophagy is involved in neurotoxicity of 6-OHDA. (A, B) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of the autophagy inhibitors 3-methyl adenine (3-MA; 4 mM), wortmannin (Wort; 100 nM), bafilomycin A1 (Baf; 2 nM), NH4Cl (10 mM) and chloroquine (Cq; 20 μM). After 24 h, cell viability was assessed by MTT (A) and LDH release test (B). (C–I) SH-SY5Y cells transfected with control or LC3β shRNA were treated with 6-OHDA (the inset in C shows immunoblot verification of LC3β knockdown). Intracellular acidification (C), phosphatidylserine externalization (F), caspase activation (G), ROS production (H) or superoxide levels (I) were examined by flow cytometry after 16 h. Cell viability was assessed by crystal violet (D) and LDH release test (E) after 24 h. The data are mean±SD values of triplicate measurements from a representative of three experiments (A, B, D, E) or mean±SD values from three independent experiments (C, F–I) (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Transfection, Control, shRNA, Western Blot, Knockdown, Activation Assay, Cytometry

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 5. AMPK/mTOR signaling is involved in neurotoxicity of 6-OHDA. (A–E) Control shRNA- or AMPK shRNA-transfected SH-SY5Y cells were treated with 6-OHDA and the cell viability was assessed by crystal violet (A) or LDH test (B) after 24 h. Phosphatidylserine externalization (C), caspase activation (D) and ROS or superoxide production (E) were examined by flow cytometry after 16 h. (F) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of rapamycin (RAP) and the cell viability was determined by MTT test after 24 h. The data are mean±SD values of triplicate measurements from a representative of three experiments (A, B, F) or mean±SD values from three independent experiments (C–E) (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, shRNA, Transfection, Activation Assay, Cytometry, Incubation

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 6. AMPK/p38 signaling is involved in neurotoxicity of 6-OHDA. (A) SH-SY5Y cells were incubated with 6-OHDA (50 μM) and the phosphorylation of p38 was assessed by immunoblotting at the indicated time points. (B, C) SH-SY5Y cells transfected with control shRNA (B, C), LC3 shRNA (B) or AMPK shRNA (C) were treated with 6-OHDA (50 μM) for 8 h and p38 activation was determined by immunoblotting. (D–F) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of p38 inhibitor SB 203580 (10 μM). Phosphorylation of p38 and AMPK, as well as LC3 conversion were assessed by immunoblot after 8 h (D), while the cell viability was determined by crystal violet staining (E) and LDH test (F) after 24 h of incubation. The representative blots are presented with the densitometry data above the relevant bands, while the data in (E, F) are mean±SD values of triplicate measurements from a representative of three experiments (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Phospho-proteomics, Western Blot, Transfection, Control, shRNA, Activation Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

doi: 10.1111/j.1582-4934.2009.00954.x

Figure Lengend Snippet: Metformin treatment activates AMPK in ovarian cancer cell lines. (A) Ovarian cancer cells (A2780, CP70, C200, SKOV3ip and PE04) were treated with metformin with indicated concentrations for 12 hrs. Protein lysates were immuno-blotted against phospho-ACC and phospho-AMPK antibodies. Same blot was stripped and probed for b-actin to show equal protein loading. (B) A2780, CP70, C200 and SKOV3ip cells were treated with indicated concentrations of metformin for 8 hrs. Cells were pulsed with 6 mM [1- 14 C] palmitic acid. After partition of cells, the upper aqueous phase was taken for measurement of radioactivity. *** P < 0.001; ** P < 0.01, * P < 0.05; NS: not significant compared to untreated cells.

Article Snippet: AMPKα 1/2 +/+ and AMPKα 1/2 –/– mefs were a kind gift from Dr. Keith R. Laderoute, (SRI International, Menlo Park, CA, USA).

Techniques: Radioactivity

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

doi: 10.1111/j.1582-4934.2009.00954.x

Figure Lengend Snippet: Metformin mediated its action via LKB1. (A) LKB null (LKB 2/2 ) and wild-type (LKB +/+ ) mefs were treated with metformin (5–10 mM) and analysed by immunoblot for AMPK and ACC phosphorylation and b actin for equal protein loading. (B) LKB 2/2 and wild-type mefs were treated with metformin with indicated concentrations for 24 hrs. After overnight fixation and propidium iodide staining cells were flow-sorted. The data are representations of two separate experiments. (C) Immunoblot analysis revealed down-regulation of LKB1 in untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected A2780 cells at 48 hrs. (D) After 48 hrs transfection, similar sets were treated with metformin for 12 hrs and processed for immunoblot for p-ACC and total ACC. (E) A2780 untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected cells were treated with indicated concentrations of metformin for 48 hrs and assessed for proliferation by MTT. ** P < 0.01, siRNA (Si) compared to C and NT.

Article Snippet: AMPKα 1/2 +/+ and AMPKα 1/2 –/– mefs were a kind gift from Dr. Keith R. Laderoute, (SRI International, Menlo Park, CA, USA).

Techniques: Western Blot, Phospho-proteomics, Staining, Control, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

doi: 10.1111/j.1582-4934.2009.00954.x

Figure Lengend Snippet: Metformin can inhibit growth independent of AMPK. (A) Immunoblot analysis revealed the down-regulation of AMPKα 1 in untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected A2780 cells at 48 hrs. Similar sets were treated with metformin (10 mM) for 12 hrs and processed for immunoblot for p-ACC and β actin (lower panel). (B) A2780 untransfected, siRNA and non-target siRNA transfected cells were treated with indicated concentrations of metformin for 48 hrs and assessed for cell number. ** P < 0.01, * P < 0.05 compared to siRNA (Si) and non-target siRNA control (NT) transfected cells. (C) AMPK null ( AMPK +/+ ) and wild-type ( AMPK +/+ ) mouse embryo fibroblast (mefs) were treated with metformin (5–10 μM) and analysed by immunoblot for pAMPK and pACC, as well as for status of proteins of mTOR pathway. (D) AMPK +/+ and wild-type mefs were treated with metformin (5–10 μM) for 72 hrs. MTT assay was performed to estimate cell viability. The data represent three separate experiments. ** P < 0.01, * P < 0.05 compared to untreated AMPK null and wild-type mefs. (E, F) AMPKα null (AMPK +/+ ) and wild-type (AMPK +/+ ) mefs were transfected with CyclinD1 and p21 reporter-luciferase constructs (0.2 μg/24 well). The next day, the cells were treated with 10 μM metformin and 24 hrs later, luciferase activity was determined using dual luciferase assay system. *** P < 0.001, ** P < 0.01 AMPK α 1/2 –/– mefs compared to AMPK α +/+ mefs (black bars). ‡ P < 0.001, † P < 0.01 between metformin treated AMPK α –/– mefs and AMPK α 1/2 +/+ mefs.

Article Snippet: AMPKα 1/2 +/+ and AMPKα 1/2 –/– mefs were a kind gift from Dr. Keith R. Laderoute, (SRI International, Menlo Park, CA, USA).

Techniques: Western Blot, Control, Transfection, MTT Assay, Luciferase, Construct, Activity Assay