Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control

Journal: Translational Psychiatry

Article Title: Synergistic inhibition of histone modifiers produces therapeutic effects in adult Shank3 -deficient mice

doi: 10.1038/s41398-021-01233-w

Figure Lengend Snippet: A , B Bar graphs showing mRNA levels of NMDAR subunits ( Grin1, Grin2a, Grin2b ) and AMPAR ( Gria1, Gria2 ) subunits ( A ) and synaptic proteins ( Shank3 , Homber1b , Arc ) ( B ) in PFC of adult saline-treated WT or Shank3 +/ΔC mice treated with saline or Rom/GSK (0.25/5 mg/kg, i.p., 3x) at 8 days post-treatment. n = 7–11 mice per group. C, D Bar graphs showing total ( C ) or synaptic ( D ) protein levels of NMDAR subunits in the 3 groups at 8 days post-treatment. C, n = 7–9 mice/group; D, n = 5–7 mice per group. Insets: representative immunoblots. In all figures, # p < 0.1, * p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: Western blots were performed using antibodies against NR1 (1:500, NeuroMab, 75‐ 272), NR2A (1:500, Millipore, 07‐632), NR2B (1:500, Millipore, 06‐600), PSD95 (1:1000, Cell Signaling, 36233 S) and tubulin (1:5000 Sigma, T9026).

Techniques: Saline, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Identification of PTGR2 inhibitors as a new therapeutic strategy for diabetes and obesity

doi: 10.1038/s44321-025-00216-4

Figure Lengend Snippet: ( A ) Metabolism of 15-keto-PGE2. ( B ) Activation of murine PPARγ (mPPARγ) measured by Gal-PPARγ/UAS-LUC reporter assay in HEK293T cells ( n = 3 per group, 3 biological replicates with 1 technical replicate each). Cells were transfected with Gal4-PPARγ, UAS-LUC, and TK-Rluc (Renilla luciferase), and treated with pioglitazone (** P = 0.0021, **** P < 0.0001, *** P = 0.0002) or 15-keto-PGE2 (** P = 0.0015, * P = 0.0137, ** P = 0.0014). RT-qPCR of ( C ) Glut4 (**** P < 0.0001, ** P = 0.0005, **** P < 0.0001) and other mPPARγ-downstream genes including ( D ) Irs2 (**** P < 0.0001, * P = 0.0475, **** P < 0.0001), ( E ) Sorbs1 (**** P < 0.0001, ** P = 0.0023), ( F ) Cd36 (*** P = 0.0002, *** P = 0.0006), ( G ) Acs (**** P < 0.0001, ** P = 0.0029), ( H ) Cepba (** P = 0.0016, * P = 0.0274, ** P = 0.0025), and ( I ) Adipoq (** P = 0.0023, **** P < 0.0001, **** P < 0.0001) in differentiated 3T3-L1 adipocytes treated with 15-keto-PGE2 ( n = 3 per group, 3 biological replicates with 2 technical replicate each). ( J ) Effect of 15-keto-PGE2 on insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes (**** P < 0.0001, *** P = 0.0002, **** P < 0.0001; n = 4 per group, 4 biological replicates with 1 technical replicate each). ( K ) HEK293T cells transfected by mPPARγ and treated with 15-keto-PGE2. Covalent binding of 15-keto-PGE2 to mPPARγ detected by liquid-chromatography tandem mass spectrometry (LC-MS/MS). ( L ) Reciprocal co-immunoprecipitation of mPPARγ and cysteine-15-keto-PGE2. Myc-DDK-mPPARγ and Myc-DDK-mPPARγ C313A were expressed in HEK293T cells, and immunoprecipitation (IP) conducted using either anti-DDK (anti-Flag) or anti-15-keto-PGE2-cysteine-BSA antibody, followed by immunoblotting with anti-15-keto-PGE2-cysteine-BSA and anti-DDK antibody. ( M ) PPRE reporter activity after addition of 15-keto-PGE2 to HEK293T cells transfected with wild-type and C313A mutant mPPARγ (** P = 0.0037, ** P = 0.0077, **** P < 0.0001, **** P < 0.0001; n = 3 per group, 3 biological replicates with 1 technical replicate each). ( N ) Native mass spectrometry spectrum showed the binding of 15-keto-PGE2 to wild-type and mPPARγ mutants (C313A and H351A). The spectrum of unbound free-form proteins was shown in the left panel. The spectrum of bound form after the addition of 15-keto-PGE2 was shown in the right panel ( n = 4 per group, 4 independent experiments with 1 technical replicate each) and ( O ) histogram (**** P < 0.0001). ( P ) 15-keto-PGE2 enhanced insulin-stimulated glucose uptake in PPARγ-null 3T3-L1 clones (#1296; n = 4 per group, 4 biological replicates with 1 technical replicate each) rescued with wild-type mPPARγ (**** P < 0.0001, ** P = 0.0036) but not in those rescued with mutant mPPARγ (C313A) (**** P < 0.0001). ( Q ) Diagram showing the motifs of mPPARγ and 15-keto-PGE2 binding site. Data information: Data are presented as mean and standard error (S.E.M.). Statistical significance was calculated by one-way analyses of variance (ANOVA) with Tukey’s post hoc test in ( B – J , P ) and two-sample independent t -test in ( M , O ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns means no statistical difference. .

Article Snippet: Murine PPARγ plasmid , OriGene, USA , Cat.# MC201042.

Techniques: Activation Assay, Reporter Assay, Transfection, Luciferase, Quantitative RT-PCR, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Activity Assay, Mutagenesis, Clone Assay

Journal: British journal of pharmacology

Article Title: Inactivation of SERCA2 Cys 674 accelerates aortic aneurysms by suppressing PPARγ.

doi: 10.1111/bph.15411

Figure Lengend Snippet: FIGURE 1 Inactivation of SERCA2 C674 suppresses PPARγ by activation of calcineurin/ NFAT/NF-κB pathways. (a) The main signalling pathways from the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis in duplicate aorta samples of WT and SKI mice in LDLR−/−background. (b) The mRNA levels of PPARγ2 in aortas of WT and SKI mice in LDLR−/−

Article Snippet: The PPARγ2 plasmids (ORIGENE, Cat# MR208132) or its control CMV-GFP plasmids (ORIGENE, Cat# PS100040) were transfected into SKI SMCs according to Effectene® Transfection Reagent Handbook (Qiagen, Cat#301305).

Techniques: Activation Assay

Journal: British journal of pharmacology

Article Title: Inactivation of SERCA2 Cys 674 accelerates aortic aneurysms by suppressing PPARγ.

doi: 10.1111/bph.15411

Figure Lengend Snippet: FIGURE 3 The down-regulation of PPARγ2 accounts for the phenotypic modulation of SKI SMCs. (a) Representative western blots from SKI aortic SMCs transfected with PPARγ2 plasmids and quantification of band intensities in graph. Data shown are means ± SEM; n = 5. *P < .05, significantly different as indicated; unpaired Student's t test.. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic SMCs. In (b–d), data shown are means ± SEM; n = 6. *P < .05, significantly different as indicated; unpaired Student's t test

Article Snippet: The PPARγ2 plasmids (ORIGENE, Cat# MR208132) or its control CMV-GFP plasmids (ORIGENE, Cat# PS100040) were transfected into SKI SMCs according to Effectene® Transfection Reagent Handbook (Qiagen, Cat#301305).

Techniques: Western Blot, Transfection, Migration