Journal: bioRxiv

Article Title: Fatty acid binding proteins shape the cellular response to activation of the glucocorticoid receptor

doi: 10.1101/2021.07.02.450968

Figure Lengend Snippet: GRE-transcriptional activity of GR in the absence or presence of FABP1, FABP2, FABP3, FABP4 or FABP5 in response to vehicle control or increasing concentrations of (A) hydrocortisone (B) dexamethasone or (C) prednisolone, was assessed using a reporter gene in COS-7 cells after 24 h treatment (n=3). (D) Expression of the GR in naïve COS-7 cells or following transfection with GR determined by immunoblotting. Transcriptional activity of the endogenous or transfected GR in the presence of FABP4 in response to (E) dexamethasone or (F) prednisolone (n=3). (G) FABP translocation to the nucleus in COS-7 cells transfected with GR and GFP-FABP1 or GFP-FABP4 following 24 h treatment with vehicle or an EC 80 concentration of hydrocortisone, dexamethasone or prednisolone (n=3). Data are mean ± SEM from n independent experiments, as stated. For concentration-response curves, symbols show means and error bars, S.E.M. *** p<0.001, two-way ANOVA with Sidak’s multiple comparison test. For bar graphs, bars show the mean, error bars the S.E.M. and symbols show the independent data points for each experiment. *** p<0.001, two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Immunoblotting used primary antibodies recognising FABP1 (Abcam (AB76812; rabbit, 1:300), FABP2 (gift from Dr Satoshi Kaiura, Dainippon Sumi-tomo Pharma Co. Ltd., Osaka, Japan; mouse; 1:400), FABP3 (R&D Systems MAB1678; rabbit, 1:200), FABP4 (Abcam AB92501; rabbit, 1:1000), FABP5 (Abcam AB37267; rabbit, 1:200), GR (Abcam AB183127; rabbit, 1:400) or β-actin (Cell Signaling Technology 3700S; mouse, 1:5000).

Techniques: Activity Assay, Control, Expressing, Transfection, Western Blot, Translocation Assay, Concentration Assay, Comparison

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: FABP3 was strongly expressed in PV+ GABAergic interneurons of the ACC. A, Left, RT-PCR analysis of Fabp3 in different brain areas. Right, qPCR analysis of Fabp3 in different brain areas. Error bar indicates mean ± SEM. n = 6 mice per group. **p < 0.01 versus WT ACC. B–F, Confocal images showing colocalization of FABP3 and a pyramidal neuronal marker (Ng), three classical GABAergic neuronal makers (PV, SOM, and CR), or GAD67 in the ACC. B, Most FABP3+ structures do not show immunoreactivity for Ng. C, Confocal images showing FABP3 and PV colocalization in the ACC. D, F, Most FABP+ structures do not show immunoreactivity for SOM (D) or CR (E). F, Confocal images showing FABP3 and GAD67 colocalization in the ACC (arrowheads). B, C (bottom), D, E (bottom right), F (right), Enlarged images of the boxed area in the merged image. Scale bars, 50 μm. Str, Striatum.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Colocalization of PV, SOM, and CR with FABP3 in the ACC a

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Fabp3 gene ablation in the ACC caused upregulation of GAD67 expression and GABA synthesis. A, Quantitative analysis of the number of PV−, SOM−, and CR+ neurons in the ACC. PV, n = 8 sections, 4 mice; SOM, n = 12 sections, 6 mice; CR, n = 12 sections, 6 mice per group. B, Representative immunoblots (left) and quantitative densitometry analysis (right) probed with GABA-related antibodies. GAD67 and β-actin, n = 6 mice per group; GAD65, VGAT, and GABAAR, n = 5 mice per group; gephyrin, n = 5 and 6 WT and Fabp3 KO mice, respectively. C, Quantitative analysis of Gad67 mRNA expression in the ACC from WT and Fabp3 KO mice. n = 4 mice per group. D, Quantitative analysis of the GABA concentrations in ACC extracts from WT and Fabp3 KO mice by ELISA. n = 6 mice per group. E–G, There were no differences in GAD67 expression or GABA synthesis in the mPFC or AM of Fabp3 KO mice. E, Representative immunoblots (left) and quantitative densitometry analysis (right) probed with GAD67 antibody. n = 4 mice per group. F, Quantitative analysis of the Gad67 mRNA expression. n = 4 mice per group. G, Quantitative analysis of the GABA concentrations in the mPFC and AM extracts by ELISA. mPFC, n = 6 mice per group; AM, n = 4 and 6 WT and Fabp3 KO mice, respectively. H, I, Quantitative analysis of the DA and 5-HT concentrations in ACC extracts by ELISA. 5-HT, n = 6 mice per group; DA, n = 6 and 5 WT and Fabp3 KO mice, respectively. A–I, **p < 0.01 versus WT mice. J, Left, Representative immunoblots probed with FABP3 antibody. Right, Quantitative analysis of Gad67 mRNA in Neuro-2a cells. n = 4 per group. **p < 0.01 versus control. mock, Mock control; OE, Fabp3 overexpression; NC, negative control; KD, Fabp3 knockdown. Error bar indicates mean ± SEM.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Negative Control

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Enhancement of inhibitory synaptic plasticity in the ACC of Fabp3 KO mice. A, Representative traces of mEPSCs and mIPSCs in ACC layer II/III pyramidal neurons. B, Plots of the mEPSC and mIPSC frequencies in WT and Fabp3 KO mice. C, Plots of the mEPSC and mIPSC amplitudes in WT and Fabp3 KO mice. n = 53 cells for WT and 59 cells for Fabp3 KO from 5 mice for mEPSCs. n = 52 cells for WT and 60 cells for Fabp3 KO from 5 mice for mIPSCs. **p < 0.01 versus WT mice. Error bar indicates mean ± SEM.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Extracellular glutamate (Glu) concentrations in the ACC in freely moving animals. A, Dialysate Glu concentrations at baseline (basal) and after depolarization stimulation (High-K+) (left). Glu AUC before (basal) and after (High-K+) depolarization stimulation (right). *p < 0.05 versus wild-type mice. B, Normalization of the dialysate Glu signals indicated that the response to depolarization stimulation was enhanced in Fabp3 KO mice (left). The normalized Glu AUC after depolarization stimulation (right). A, B, Error bar indicates mean ± SEM. *p < 0.05 versus WT mice. n = 6 WT mice; n = 5 Fabp3 KO mice. C, D, Representative immunoblots (left) and quantitative densitometry analysis (right) probed with various antibodies. *p < 0.05; **p < 0.01 versus WT mice. n = 6 mice per group. Error bar indicates mean ± SEM.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Decreased binding of MeCP2 and HDAC1 to the Gad67 promoter region in the ACC of Fabp3 KO mice. A, Representative immunoblots (top) and quantitative densitometry analysis (bottom) probed with various antibodies are shown. n = 6 mice per group. B, ChIP assay of ACC extracts from WT or Fabp3 KO mice via anti-MeCP2 and HDAC1. The precipitated DNA was analyzed by qPCR with primers amplifying the Gad67 promoter region. **p < 0.01 versus WT mice. n = 5 WT mice. n = 4 Fabp3 KO mice. C, DNA methylation profile in the Gad67 promoter region determined by bisulfite sequencing. Black circles represent methylated CpGs. Open circles represent unmethylated sites. Top numbers indicate global percentages of methylated cytosines. D, Quantitative analysis of the Gad67 mRNA expression in the ACC. WT, n = 6; KO, n = 5; KO + MET, n = 6 mice per group. **p < 0.01 versus WT mice. ##p < 0.01 versus KO mice. E, F, Quantitative analysis of the SAM concentration by ELISA. n = 4 per group. **p < 0.01 versus mock control. MET treatment (5.2 mmol/kg, s.c., twice per day for 6 d). mock, Mock control; OE, Fabp3 overexpression. Error bar indicates mean ± SEM.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: Binding Assay, Western Blot, DNA Methylation Assay, Methylation Sequencing, Methylation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Amelioration of behavioral abnormalities in Fabp3 KO mice by chronic treatment with MET. A, Schematic of the behavioral tests and MET treatment (5.2 mmol/kg, s.c., twice per day). During the behavioral testing period, mice received saline or MET once per day after testing. B, OFT. Graph represents the time spent in the center area over a 5 min period for WT and Fabp3 KO mice. n = 14–17 mice per group. C, HBT. An illustrative example of the travel pathway in the HBT using video tracking software (top). Graphs represent the time spent in the center area, total number of head-dips, head-dip duration, and latency to first head-dip for the 5 min testing session (bottom). n = 13–15 mice per group. **p < 0.01 versus saline-treated WT mice. ##p < 0.01 versus saline-treated KO mice. D, NORT. Differences in exploratory preference were assessed between groups in the training (left) or test (right) sessions. n = 11–15 mice per group. **p < 0.01 versus familiar group. Sal, Saline treatment. MET treatment (5.2 mmol/kg, s.c., twice per day for 6 d). Error bar indicates mean ± SEM.

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: Software

Journal: The Journal of Neuroscience

Article Title: FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase 67 Promoter Region

doi: 10.1523/JNEUROSCI.1285-18.2018

Figure Lengend Snippet: Locomotor activities during behavioral tests a

Article Snippet: The primary antibodies included mouse monoclonal antibodies against FABP3 (1:200, Hycult Biotechnology HM2016, RRID: AB_533050 ); rabbit polyclonal antibodies against neurogranin (Ng, 1:2000, Millipore AB5620, RRID: AB_91937 ); rabbit polyclonal antibodies against PV (1:2000, Abcam ab11427, RRID: AB_298032 ); rat monoclonal antibodies against somatostatin (SOM, 1:50, Millipore MAB354, RRID: AB_2255365 ); and rabbit polyclonal antibodies against calretinin (CR, 1:2000, Swant CR7697, RRID: AB_2619710 ).

Techniques: