Journal: Biology of Sex Differences

Article Title: Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

doi: 10.1186/s13293-021-00394-z

Figure Lengend Snippet: Antibodies

Article Snippet: AMPKα1 , Santa Cruz , sc19126 , 1/1000.

Techniques:

Journal: Biology of Sex Differences

Article Title: Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

doi: 10.1186/s13293-021-00394-z

Figure Lengend Snippet: Cardiac-specific Ampkα2 inactivation 16 weeks after tamoxifen injection in adult male and female mice. A Protein content of AMPKα2 and AMPKα1 in left ventricle (LV) homogenates. Tubulin is used as a loading control. B Immunoblotting of total AMPK (tAMPK), total ACC (tACC), phosphorylated-AMPK (pAMPK) and phosphorylated-ACC (pACC) in LV. Tubulin is used as a loading control. C Protein content of AMPKα2 and AMPKα1 in skeletal muscle. Actin is used as a loading control. ( n = 3 to 4 per experimental group). ANOVA: § p ≤ 0.05, §§ p ≤ 0.01, §§§ p ≤ 0.001 for the genotype effect. Post hoc Tukey test: * p < 0.05, *** p < 0.001 Ampkα2 f/f vs Ampkα2ciKO

Article Snippet: AMPKα1 , Santa Cruz , sc19126 , 1/1000.

Techniques: Injection, Control, Western Blot

Journal: Scientific Reports

Article Title: Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway

doi: 10.1038/s41598-017-03477-3

Figure Lengend Snippet: AMPK activation is required for OMS-induced phagosomal maturation and antimicrobial responses. ( a and b ) BMDMs were treated with OMS-A (10 μM; for a ) or OMS-B (10 μM; for b ) for indicated times (0–18 h). The cell lysates were subjected to immunoblot analysis of p-AMPKα, p-ACC, and Actin. The entire blots are shown in Supplementary Fig. . The densitometric values for p-AMPKα were normalized to Actin ( bottom ). ( c and d ) BMDMs were transduced with non-specific shRNA (sh NS ) or Ampk -specific shRNA (sh Ampk )-expressing lentivirus for 48 h and then infected with ERFP-Mtb (moi = 10) for 4 h, followed by treatment with OMS-A or OMS-B for 24 h. ( c and d ) Mtb-ERFP (red), Alexa 488-conjugated-LC3 (green), and DAPI (blue) were detected by confocal analysis. Scale bar, 5 µm. ( c ) Representative confocal microscopic images from three independent samples are shown. ( d ) Quantitative analysis of cells showing the colocalization between LC3 and Mtb-ERFP. For each experiment, at least 100 cells were scored from 6 random fields. ( e ) After 3 days of infection, intracellular bacterial loads were determined by CFU assay. (inset) RT-PCR analysis of Ampk mRNA expression of transduction efficiency. Data shown are from one representative of at least three independent experiments (means ± SD of triplicates [ a , b bottom ; d ] samples]). *p < 0.05, ***p < 0.001, compared with sh NS . U, uninfected/untreated; SC, solvent control.

Article Snippet: The pLKO.1-based target shRNA plasmids for m Ampkα (Santa Cruz Biotechnology, sc-29674-SH) or h ATG5 (Santa Cruz Biotechnology, sc-41445-SH) were cotransfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, 12566014, Carlsbad, CA, USA) for 72 h. Then, the lentivirus-containing supernatant was collected, filtered, and titrated.

Techniques: Activation Assay, Western Blot, Transduction, shRNA, Expressing, Infection, Colony-forming Unit Assay, Reverse Transcription Polymerase Chain Reaction, Solvent, Control

Journal: Scientific Reports

Article Title: Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway

doi: 10.1038/s41598-017-03477-3

Figure Lengend Snippet: OMS inhibits Mtb-induced inflammatory responses through AMPK activation. ( a ) BMDMs were infected with Mtb (moi = 10) for 4 h and then treated with OMS-A (1, 5, 10 μM) or OMS-B (1, 5, 10 μM) for 24 h. ( b ) BMDMs were transduced with adenoviral NF-ĸB-luciferase reporter plasmid for 36 h, and infected with Mtb and then treated with OMS-A or OMS-B for 6 h. The cells were harvested and NF-ĸB luciferase reporter activity was determined. ( c ) BMDMs were transduced with non-specific shRNA (sh NS ) or Ampk -specific shRNA (sh Ampk )-expressing lentivirus for 48 h and then infected with Mtb, followed by treatment with OMS-A or OMS-B for 24 h. ( a and c ) The supernatants were harvested and subjected of ELISA analysis of TNF-α, IL-6, IL-1β, and IL-12p40 production. All data represent the means ± SD of triplicates from each sample. **p < 0.01, ***p < 0.001, compared with SC ( a – c ). U, uninfected/untreated; SC, solvent control; ns, no significant.

Article Snippet: The pLKO.1-based target shRNA plasmids for m Ampkα (Santa Cruz Biotechnology, sc-29674-SH) or h ATG5 (Santa Cruz Biotechnology, sc-41445-SH) were cotransfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, 12566014, Carlsbad, CA, USA) for 72 h. Then, the lentivirus-containing supernatant was collected, filtered, and titrated.

Techniques: Activation Assay, Infection, Transduction, Luciferase, Plasmid Preparation, Activity Assay, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Solvent, Control

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