Journal: Molecular Neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: RT-PCR primers

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Sequencing

Journal: Molecular Neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: Differential expression and functional analysis in astrocytes overexpressing circKat6b . A Volcano map of DEGs in the pCDH5-circKat6b vs pCDH5-copGFP where red represents upregulated genes and blue represents downregulated genes. B GO (CC, MF, BP) classification, C KEGG pathway analysis, and D reactome enrichment analysis of DEGs related to circKat6b overexpression. E RT-PCR results of GABAB1 , stat1 , and circKat6b mRNA in C8D1A overexpressing circKat6b . F Western blot results of GABAB1, stat1, and p-stat1 in C8D1A overexpressing circKat6b . N = 4, vs pCDH5-copGFP group, * P < 0.05, ** P < 0.01 using Student’s t -test

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Quantitative Proteomics, Functional Assay, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: CircKat6b regulates astrocyte function via stat1. A Protein expression of stat1 and p-stat1 protein in the hippocampus in each group. N = 6, vs control group, ** P < 0.01; vs CUMS group, # P < 0.05 using one-way ANOVA followed by Tukey’s test. B Effect of circKat6b overexpression on stat1 and p-stat1 protein expression in the hippocampus in each group. N = 6, vs control-circCon group, ** P < 0.01; vs control-circKat6b group, $$ P < 0.01; vs CUMS-circKat6b group, ## P < 0.01; vs CUMS + Es-circCon group, && P < 0.01 using two-way ANOVA followed by Tukey’s test. C Representative double-staining immunofluorescence of p-stat1 and GFAP in brain sections from different groups following esketamine treatment. Scale bar = 20 µm

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Expressing, Control, Over Expression, Double Immunofluorescence Staining

Journal: Molecular Neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: A schematic diagram depicting how esketamine affects the function of astrocytes through the regulation of stat1 expression via circKat6b to exert an antidepressant effect

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Human cytomegalovirus protein UL42 antagonizes cGAS/MITA-mediated innate antiviral response

doi: 10.1371/journal.ppat.1007691

Figure Lengend Snippet: (A) HCMV UL42 inhibits cGAS-MITA-induced IFNβ promoter and ISRE activation in a dose-dependent manner. HEK293T/MITA cells (1x10 5 ) were transfected with the IFNβ promoter (0.05 μg) or ISRE (0.03 μg) reporter plasmid, and expression plasmids for cGAS (0,01 μg) and increased amounts of UL42 (0, 0.025, 0.05, and 0.1 μg) for 20 hrs before luciferase assays. (B) Effects of UL42 on IFN-β-induced STAT1/2 activation. HEK293 cells (1x10 5 ) were transfected with STAT1/2 reporter (0.005 μg) and UL42 expression (0.05 μg) plasmids for 20 hrs. The cells were then untreated or treated with IFN-β for 12 hrs before luciferase assays. (C) HCMV UL42 inhibits HCMV-, HSV-1-, and VACV-induced transcription of antiviral genes in HFFs. UL42-stable HFFs (4x10 5 ) were un-infected or infected with HCMV (MOI = 1), HSV-1 (MOI = 1), or VACV (MOI = 1) for the indicated times (upper histographs) or 12 h (lower histographs) before qPCR analysis. The immunoblots show the expression levels of UL42 in the HFF-UL42 stable cell lines. (D) HCMV UL42 inhibits dsDNA-induced transcription of antiviral genes in HFFs. UL42 stable HFFs (4x10 5 ) were transfected with HSV120 (2 μg), DNA90 (2 μg), VACV70 (2 μg), or ISD (2 μg) for the indicated times before qPCR analysis. (E) UL42 impairs HCMV- and HSV120-induced phosphorylation of downstream components. UL42 stable HFFs (4x10 5 ) were infected with HCMV (MOI = 1) or transfected with HSV120 (2 μg/ml) for the indicated times before immunoblot analysis. The lower panels are results of qPCR analysis for HCMV UL123 mRNA or HSV120 DNA. (F) Effect of UL42 on IFN-β-induced phosphorylation of STAT1. UL42 stable HFFs (4x10 5 ) were untreated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblot analysis. Graphs show mean ± SD, n = 3. *p<0.05, **p<0.01 (unpaired t test).

Article Snippet: 2’ 3’-cGAMP, and lipofectamine 2000 (Invitrogen); polybrene (Millipore); puromycin and RNase inhibitor (Thermo); dual-specific luciferase assay kit (Promega); SYBR (BIO-RAD); digitonin (Sigma); streptavidin agarose (Solulink); mouse antibodies against Flag, and β-actin (Sigma), and HA (Covance); rabbit monoclonal antibodies against cGAS (66546S/31659S), MITA (13647S), phosphor-MITA (85735S), phosphor-p65, and phosphor-IRF3 (4947S) (Cell Signaling Technology), phosphor-TBK1(ab109272) and TBK1(ab40676) (Abcam), IRF3 (sc-9082), phosphor-Tyrosine701-STAT1(9167S) and STAT1(sc-346) (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Infection, Western Blot, Stable Transfection