Journal: PLoS ONE

Article Title: Adenylate kinase 1 overexpression increases locomotor activity in medaka fish

doi: 10.1371/journal.pone.0257967

Figure Lengend Snippet: (A) Phylogenetic tree showing the relationship of the AK isozymes of human, mouse and medaka. (B) Electrophoresis image of Ak1 and Actb RT-PCR products in various adult medaka tissues. B, Brain; E, Eye; O, Ovary; L, Liver; S, Skin; N, No-template control. (C) Structure of Ak1 overexpression construct. The construct consisted of the medaka Actb regulatory region, Ak1 cDNA (without the stop codon), C-terminus 3× FLAG tag, internal ribosome entry site (IRES), green fluorescent protein (GFP) open reading frame, and SV40 polyA signal. (D) Design of genetic crosses for generating heterozygous Ak1 -overexpressing ( Ak1 -OE) medaka and their wild-type (WT) siblings. (E) Bright-field and fluorescence images of WT and Ak1 -OE embryos at 5 days post-fertilization (dpf). (F) Ak1 expression level of larvae at 9 dpf. Data represent the mean ± SEM, n = 4 each. WT expression level was set at 1. The p -value was calculated using two-tailed Welch’s t -test. Each dot represents an individual value. (G) Western blotting for Ak1 (left) and FLAG tag (right). Black and white arrows indicate Ak1-FLAG and Ak1, respectively.

Article Snippet: Ak1 polyclonal primary antibody (1:500) (14978-1-AP; Proteintech, Rosemont, IL, USA) was dissolved in 5% skim milk in TBST and incubated for 1 h at room temperature.

Techniques: Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Control, Over Expression, Construct, FLAG-tag, Fluorescence, Expressing, Two Tailed Test, Western Blot

Journal: PLoS ONE

Article Title: Adenylate kinase 1 overexpression increases locomotor activity in medaka fish

doi: 10.1371/journal.pone.0257967

Figure Lengend Snippet: (A) Structure of the behavioral assay. Larvae (8 dpf) were placed into a photoperiod- and temperature-controlled chamber (14L10D, 25°C) at the end of the light phase. After 10 h of habituation (Hab.), recording was started at the onset of the light phase at 9 dpf and continued for 3 full days. (B) Mean locomotor activity of Ak1 -overexpressing ( Ak1 -OE) and wild-type (WT) larvae. (C) Total locomotor activity of Ak1 -OE and WT larvae per day. Data represent the mean ± SEM and were analyzed via two-way repeated measures ANOVA. Effect of time, F (1.386, 37.43) = 11.45, p = 0.0006; effect of overexpression, F (1, 27) = 7.891, p = 0.0091; effect of interaction, F (2, 54) = 0.2186, p = 0.8044. * p < 0.05; n.s., not significant (Dunnett’s multiple comparisons test, vs. day 1). (D) Total locomotor activity of Ak1 -OE and WT larvae per day during the light phase (white) and dark phase (gray). Data represent the mean ± SEM. The p -values (* p < 0.05) were calculated using two-tailed Welch’s t -test. Each dot represents an individual value. (B–D) n = 15 (WT), n = 14 ( Ak1 -OE).

Article Snippet: Ak1 polyclonal primary antibody (1:500) (14978-1-AP; Proteintech, Rosemont, IL, USA) was dissolved in 5% skim milk in TBST and incubated for 1 h at room temperature.

Techniques: Behavioral Assay, Activity Assay, Over Expression, Two Tailed Test

Journal: PLoS ONE

Article Title: Adenylate kinase 1 overexpression increases locomotor activity in medaka fish

doi: 10.1371/journal.pone.0257967

Figure Lengend Snippet: (A) Structure of the behavioral assay. Larvae were maintained in a chamber under a 14L10D cycle at 15°C. (B) Mean locomotor activity of Ak1 -overexpressing ( Ak1 -OE) and wild-type (WT) larvae. (C) Total locomotor activity of Ak1 -OE and WT larvae per day. Data represent the mean ± SEM and were analyzed via two-way repeated measures ANOVA. Effect of time, F (1.910, 68.74) = 1.848, p = 0.1669; effect of overexpression, F (1, 36) = 3.607×10 −5 , p = 0.9952; effect of interaction, F (2, 72) = 0.2813, p = 0.7556. n.s., not significant (Dunnett’s multiple comparisons test, vs. day 1). (D) Total locomotor activity of Ak1 -OE and WT larvae per day during the light phase (white) and dark phase (gray). Data represent the mean ± SEM. n.s., not significant (two-tailed Welch’s t -test). Each dot represents an individual value. (B–D) n = 20 (WT), n = 18 ( Ak1 -OE).

Article Snippet: Ak1 polyclonal primary antibody (1:500) (14978-1-AP; Proteintech, Rosemont, IL, USA) was dissolved in 5% skim milk in TBST and incubated for 1 h at room temperature.

Techniques: Behavioral Assay, Activity Assay, Over Expression, Two Tailed Test

Journal: Cell Research

Article Title: Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress

doi: 10.1038/s41422-019-0163-6

Figure Lengend Snippet: a AMPKγ1-D317A impairs glucose-starvation-induced AMPK activation. HA-tagged AMPK-γ1 and its D317A mutant were re-introduced into AMPKγ-TKO HEK293T cells. Cells were then deprived of glucose for 2 h, followed by immunoblotting. b, c AMPKγ2-R531G blocks the phosphorylation of cytosolic and mitochondrial AMPK under moderate and high AMP levels. HA-tagged AMPK-γ2 and its R531G mutant were re-introduced into AMPKγ-TKO HEK293T cells. Cells were then deprived of glucose for 2 h, followed by fractionation and immunoblotting for analyzing p-AMPKα (b), or by immunoprecipitation and immunoblotting for analyzing p-ACC1 and p-ACC2 (c). Statistical analysis data were shown in mean ± SD; ***p < 0.001, *p < 0.05, N.S., not significant by ANOVA, n = 3. d Knockout of AK1 significantly dampens the activation of mitochondrial- and cytosolic-localized AMPK in high AMP conditions. MEFs with AK1 being knocked out were deprived of glucose or both glucose and glutamine for 2 h, followed by fractionation and immunoblotting for analyzing p-AMPKα. Statistical analysis data were shown in mean ± SD; ***p < 0.001, **p < 0.01, *p < 0.05, N.S., not significant by ANOA, n = 3. e A simplified model depicting that the differentially compartmentalized pools of AMPK are activated with different dependencies on AXIN, and the severities of nutrient or energy stress. Glucose starvation, without increase of AMP levels, exclusively activates the lysosomal pool of AMPK through the AXIN-based pathway (➊) which phosphorylates substrates including ACC1, SREBP1, TSC2, Raptor, HDAC4, ULK1, and TBC1D1 to elicit early anti-anabolic roles. Moderately increased AMP levels, during the early phase of severe starvation or after treatment of low concentrations of AICAR, activates cytosolic AMPK, in addition to the lysosomal AMPK (➋), still dependent on AXIN. When AMP levels go up further as a result of severe starvation, ischemia, or treatment of high concentrations of AICAR, cytosolic AMPK (➌) and mitochondrial AMPK (➍) are activated independently of AXIN, leading to phosphorylation of ACC2 and MFF and accelerating catabolic activities, along with all the other substrates that can be phsophorylated at lower AMP levels. Of note, TSC2 and Raptor can be phosphorylated by all the four modes to respectively inhibit mTORC1 activity. Experiments in this figure were performed three times. See also Supplementary information, Fig. S4

Article Snippet: Rabbit anti-AK1 (cat. 14978-1-AP, 1:500 for IB) and rabbit anti AXIN2 (cat. 20540-1-AP, 1:1000 for IB, only for Supplementary information , Fig. S1h ) were purchased from Proteintech.

Techniques: Activation Assay, Mutagenesis, Western Blot, Fractionation, Immunoprecipitation, Knock-Out, Activity Assay