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: Oncogene

Article Title: Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cells by targeting the transcription factors FOXO3a and FOXM1

doi: 10.1038/s41388-017-0032-z

Figure Lengend Snippet: AMPK suppresses FOXM1 expression in HNSCC cells. a Representative images of FOXM1 IHC staining in mouse tongue tumors derived from PCI-13 stable cell lines. b Quantitation of nuclear FOXM1 staining of a . ** p < 0.01. c Western blot analysis of CRISPR-Cas9/AMPKα1 KO UM-SCC-1 cell lines

Article Snippet: AMPKα1 and FOXO3a CRISPR/Cas9 knockout (KO) plasmids sc-400104 and sc-400308 were from Santa Cruz Technologies (Dallas, TX, USA).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Stable Transfection, Quantitation Assay, Staining, Western Blot, CRISPR

Journal: Epigenetics & Chromatin

Article Title: Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation

doi: 10.1186/s13072-019-0281-x

Figure Lengend Snippet: Knock-out of AMPK caused severe differentiation defect in C2C12 cells. a The strategy for knocking-out (KO) AMPKa1 ( Prkaa1 ) and a2 ( Prkaa2 ) in C2C12 cells. For knocking-out AMPKa1 ( Prkaa1 ), CRISPR/Cas9 knockout plasmid and homology-directed repair (HDR) plasmid from Santa Cruz were used. For knocking-out AMPKa2 ( Prkaa2 ), a sgRNA targeting exon 4 of AMPKa2 ( Prkaa2 ) was used. Complete KO of AMPKa1 and a2 was confirmed by sequencing. The location of the two-base pair (TT) insertion in exon 2 of Prkaa1 and deletions in exon 4 of Prkaa2 alleles are indicated (purple dashed lines). b Knockout of AMPK in C2C12 cells was confirmed by Western blot analysis. GAPDH was used as a control. Shown are three independent clones of AMPK-KO. c , d Differentiation defects of AMPK-KO C2C12 cells. C2C12 cells were subjected to differentiation in 2% horse serum. MHC (Myosin heavy chain 1, MYH1) was detected by Western blot analysis ( c ) and immunofluorescence staining ( d ) during differentiation. Proteins examined are indicated. DAPI: 4′,6-diamidino-2-phenylindole. e Knockout of AMPK does not significantly change the expression of Tet1 , Tet2 and Tet3 at mRNA levels in C2C12 cells. Expression data were retrieved from RNA sequencing data. TPM: transcripts per million. f Gene ontology analysis of downregulated genes in AMPK-KO C2C12 cells compared to wild-type cells at myoblast (differentiation d0) and myotube (differentiation d8) stages. g The expression of Pax7 and myogenic regulatory factors (MRFs) was examined by RT-qPCR analysis. mRNA levels are presented relative to the levels in wild-type myoblasts (differentiation day 0) and were normalized to those of Gapdh . Data are presented as mean ± SD from three independent experiments performed in triplicates. * p < 0.05; ** p < 0.01

Article Snippet: For knocking-out AMPKα1 ( Prkaa1 ), CRISPR/Cas9 knockout plasmid (sc-430618) and homology-directed repair (HDR) plasmid (sc-430618-HDR) from Santa Cruz were used.

Techniques: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Western Blot, Clone Assay, Immunofluorescence, Staining, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: Epigenetics & Chromatin

Article Title: Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation

doi: 10.1186/s13072-019-0281-x

Figure Lengend Snippet: S97E mutation of TET2 partly rescues the differentiation defect of the AMPK-/- C2C12 cells. a The strategy for knocking in (KI) the p.S97E mutation of TET2 in AMPK-/- C2C12 cells. Upper panel: the target region. Lower panel: the HDR (homology-directed repair) donor. The c.289A > G;290G > A;291T > G mutation (red line) was introduced into the 3′ arm of exon 3 of the mouse TET2 gene to encode the phosphor mimic (serine-97-glutamic acid, S97E) of TET2. b Gene ontology (GO) analysis of differentially expressed genes (DEGs) between AMPK-/-:FLAG-BirA-TET2-S97E (TET2-S97E) and AMPK-/-:FLAG-BirA-TET2-WT (TET2-WT) C2C12 cells. c Myod1 , Myog and Pax7 mRNA contents were detected by RT-qPCR. Results of two independent C2C12 clones of AMPK-/-:FLAG-BirA-TET2-S97E (TET2-S97E) and AMPK-/-:FLAG-BirA-TET2-WT (TET2-WT) are shown. d Increased myosin heavy chain (MHC) expression after eight days induction of differentiation in AMPK-/-:FLAG-BirA-TET2-S97E (TET2-S97E) cells compared to AMPK-/-:FLAG-BirA-TET2-WT (TET2-WT) cells. Representative Western blot results are shown

Article Snippet: For knocking-out AMPKα1 ( Prkaa1 ), CRISPR/Cas9 knockout plasmid (sc-430618) and homology-directed repair (HDR) plasmid (sc-430618-HDR) from Santa Cruz were used.

Techniques: Mutagenesis, Quantitative RT-PCR, Clone Assay, Expressing, Western Blot