Journal: Cell Research
Article Title: Raptin, a sleep-induced hypothalamic hormone, suppresses appetite and obesity
doi: 10.1038/s41422-025-01078-8
Figure Lengend Snippet: a Schematic diagram illustrating the injection of AAV-Grm3-Flp-EGFP into PVN of control and PVN-specific Grm3 -deficient male mice to label PVN GRM3 neurons, followed by neuronal activity recording of PVN GRM3 neurons in brain slice before and during Raptin treatment. Raptin was dissolved in ACSF at a concentration of 1 ng/mL and incubated with brain slices for 10 min. b , c Representative traces ( b ) and action potential frequency ( c ) of PVN GRM3 neurons from control and PVN-specific Grm3 -deficient male mice ( n = 5 per group). d Representative images of c-Fos (green) in the GRM3 + neurons of the stomach from control and stomach-specific Grm3 -deficient male mice followed by administration of Raptin via tail vein at a dose of 1 mg/kg body weight for 1 h. Scale bar, 50 μm. e Representative images of labeled mitochondria (cyan) and KHC (red) in primary hypothalamus neurons transfected with control or Grm3 siRNA, followed by treatment of Raptin or PBS. These neurons were transfected with Mito-BFP and Khc-RFP plasmids to label mitochondria and KHC protein, respectively. Scale bar, 5 μm. f Quantification of the fluorescent KHC present on mitochondria, defined by the intensity of KHC in the region overlapping Mito-BFP ( n = 4 per group). g KEGG analysis of changed phosphorylation in the hypothalamic GT1-7 neurons through global quantitative phosphoproteomic analysis. Hypothalamic GT1-7 neurons were treated with PBS or Raptin at 10 ng/mL for 1 h. h Representative western blot of AKT signaling in cell lysates and KHC expression in mitochondria enriched from primary neurons treated with Raptin or Grm3 siRNA. Raptin was used at 10 ng/mL for 1 h. CYTO1 was used as the internal control of mitochondria. i Representative western blot of AKT signaling in cell lysates and KHC expression in mitochondria enriched from primary neurons treated with Raptin or PI3K-AKT pathway inhibitors (Wortmannin and LY294002). CYTO1 was used as the internal control of mitochondria. j , k Representative images ( j ) and quantification ( k ) of the fluorescent KHC (red) present on mitochondria, defined by the intensity of KHC in the region overlapping Mito-BFP (cyan) in primary hypothalamus neurons. Scale bar, 5 μm ( n = 4 per group). l , m Schematic diagram illustrating the injection of AAV-hSyn-Ctrl or AAV-hSyn-sh Khc in the PVN of 2-month WT male mice to generate the control and PVN-specific Khc knockdown mice, followed by treatment of PBS or Raptin. Representative immunofluorescence images ( l ) and quantification ( m ) of c-Fos (red) expression in PVN. Scale bar, 50 μm. n 24-h food intake of the PVN-specific Khc knockdown mice and control mice with or without treatment of Raptin ( n = 4 per group). Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, ***/### P < 0.001 by a two-tailed paired Student’s t -test ( c ) or by two-way ANOVA ( f , k , m , n ). See also Supplementary information, Figs. – .
Article Snippet: Grm3 flox/flox mice (Cat# S-CKO-00640), Cas9-EGFP[KI/+] mice (Cat# C001475), Pomc -Cre mice (Cat# C001448) and AgRP -Cre mice (Cat# C001249) were purchased from Cyagen Bioscience.
Techniques: Injection, Control, Activity Assay, Slice Preparation, Concentration Assay, Incubation, Labeling, Transfection, Phospho-proteomics, Western Blot, Expressing, Knockdown, Immunofluorescence, Two Tailed Test
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