Journal: The Journal of Neuroscience

Article Title: Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease

doi: 10.1523/jneurosci.1333-22.2023

Figure Lengend Snippet: Figure 3. Effects of HHcy diet on CN/NFAT4 properties. A, Confocal micrographs of the hippocampal CA1 region showing strong colocalization of NFAT4 (green) with GFAP-positive (red) astrocytes. Right, Note in the merged image that NFAT4 colocalizes to the nucleus of many GFAP-positive astrocytes, indicating increased NFAT4 activation. Scale, 50mm. B, Strong nuclear localization of NFAT4 is highlighted in an astrocyte magnified and 3D rendered from the merged image in A (right, hatched box). Bottom, The same astrocyte at higher magnification. Calibration, 2 mm) and rotated to emphasize the nuclear colocalization of NFAT4 (in teal). C, Representative EMSA from brain tissue harvested from CT diet and HHcy diet mice illustrate NFAT/ DNA binding activity. The arrowhead points to bands that are sensitive (i.e., exhibit a block shift) to the addition of a mononclonal NFAT4 antibody (Extended Data Fig. 3-1). D, NFAT4/DNA binding activity was significantly increased in HHcy diet mice. E, Representative WB for the CN Aa subunit in cytosolic and nuclear fractions harvested from brains of CT diet and HHcy diet mice. Note that using an N-terminal antibody, we observed a single band ;60 kDa in both diet groups, which represents the FL form of CN A. No signs of proteolysis (i.e., bands in the 45–57 kDa range) related to constitutively high CN activity were observed. F, In both diet groups, CN levels were highest in cytosolic fractions, and the nuclear/cytosolic ratio was not affected by diet; n = 4–5 mice/group in D and F. Significance determined with unpaired t tests. G, Representative two-photon images showing Gfa2-dependent EGFP expression in barrel cortex astrocytes of individ- ual mice injected with AAV-Gfa2-EGFP or AAV-Gfa2-VIVIT-EGFP vectors. Extended Data Figure 3-2 shows that EGFP volume increases progressively with time on HHcy diet. Images in G were taken at 3 months postdiet. Scale bar, 30 mm. H, EGFP volume in AAV-Gfa2-EGFP or AAV-Gfa2-VIVIT-EGFP-treated mice at the prediet time point (pre) and after 3 months (m) of CT or HHcy diet (3months). GFAP promoter-driven EGFP expression in HHcy mice is significantly increased over prediet baseline levels. No significant changes in EGFP levels were observed across time points in CT diet mice or in HHcy mice treated with VIVIT. In D, F, H each individual data point represents an individual mouse (n = 12–13 mice/group). Significance determined with rmANOVA followed by paired t tests (pre vs 3m), within diet group.

Article Snippet: Primary antibodies were applied overnight at 4°C (1:75 NFAT4; catalog #SC-8405 Santa Cruz Biotechnology; 1:200 GFAP; catalog #12389S, Cell Signaling Technology), diluted to proper concentration using blocking buffer.

Techniques: Activation Assay, Binding Assay, Activity Assay, Blocking Assay, Expressing, Injection

Journal: Cellular & Molecular Biology Letters

Article Title: Pou3f1 mediates the effect of Nfatc3 on ulcerative colitis-associated colorectal cancer by regulating inflammation

doi: 10.1186/s11658-022-00374-0

Figure Lengend Snippet: Pou3f1 physically interacted with Nfatc3, and it was upregulated in colons of UC-CRC mice. (A) The schematic diagram for animal model protocol. (B-C) The colon length in mice was recorded. (D) Representative images of HE-stained colon sections. (E) Nfatc3 mRNA levels in colons were determined using qPCR. (F) Western blot analysis for Nfatc3 protein levels in colons and quantitative results. (G-H) ChIP-PCR and ChIP-qPCR were performed to determine the interaction between Nfatc3 and Pou3f1 in colons. M: Marker. (I) ChIP-qPCR assay showed that Nfatc3 bound to the Src promoter, rather than the Cdx2 promoter in colon tissues. Src was used as a positive control. Cdx2 was used as a negative control. (J) The relative mRNA levels of Pou3f1 were detected by qPCR analysis. Data were from n = 6 mice per group. Values were mean ± SD. **, p < 0.01

Article Snippet: Membranes were incubated with primary antibodies, including Nfatc3 antibody (1:1000 dilution; 18222-1-AP, Proteintech), Pou3f1 antibody (1:1000 dilution; A19330, ABclonal), iNOS antibody (1:1000 dilution; A0312, Abclonal), COX-2 antibody (1:1000 dilution; A1253, Abclonal) and GAPDH antibody (1:10000 dilution; 60004-1-Ig, Proteintech).

Techniques: Animal Model, Staining, Western Blot, ChIP-qPCR, Marker, Positive Control, Negative Control

Journal: Cellular & Molecular Biology Letters

Article Title: Pou3f1 mediates the effect of Nfatc3 on ulcerative colitis-associated colorectal cancer by regulating inflammation

doi: 10.1186/s11658-022-00374-0

Figure Lengend Snippet: Pou3f1 was identified as a direct transcriptional target of Nfatc3. (A) The schematic of the potential motifs and binding sites of Nfatc3 in the Pou3f1 promoter region. (B-C) ChIP-PCR and ChIP-qPCR were performed to demonstrate the direct interaction between Nfatc3 and Pou3f1 in RAW264.7 cells. M: Marker. (D) ChIP-qPCR assay demonstrated that Nfatc3 bound to the Src promoter, rather than the Cdx2 promoter in RAW264.7 cells. Src was used as a positive control. Cdx2 was used as a negative control. (E) The relative luciferase activity was measured by luciferase reporter assay in RAW264.7 cells. (F) The mRNA levels of Pou3f1 in RAW264.7 cells and BMDMs were measured by qPCR. (G) Western blot analysis and quantification results for Pou3f1 protein levels in RAW264.7 cells and BMDMs. n = 3. Values were mean ± SD. *, p < 0.05; ** p < 0.01

Article Snippet: Membranes were incubated with primary antibodies, including Nfatc3 antibody (1:1000 dilution; 18222-1-AP, Proteintech), Pou3f1 antibody (1:1000 dilution; A19330, ABclonal), iNOS antibody (1:1000 dilution; A0312, Abclonal), COX-2 antibody (1:1000 dilution; A1253, Abclonal) and GAPDH antibody (1:10000 dilution; 60004-1-Ig, Proteintech).

Techniques: Binding Assay, ChIP-qPCR, Marker, Positive Control, Negative Control, Luciferase, Activity Assay, Reporter Assay, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Pou3f1 mediates the effect of Nfatc3 on ulcerative colitis-associated colorectal cancer by regulating inflammation

doi: 10.1186/s11658-022-00374-0

Figure Lengend Snippet: Pou3f1 was required for Nfatc3-induced inflammation in LPS-treated macrophages. (A) The relative mRNA levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by qPCR. (B) The protein levels of iNOS and COX-2 were detected using Western blot. (C-D) Flow cytometry analysis and quantification results for the fluorescence intensity of ROS. n = 3. Values were mean ± SD. *, p < 0.05; **, p < 0.01

Article Snippet: Membranes were incubated with primary antibodies, including Nfatc3 antibody (1:1000 dilution; 18222-1-AP, Proteintech), Pou3f1 antibody (1:1000 dilution; A19330, ABclonal), iNOS antibody (1:1000 dilution; A0312, Abclonal), COX-2 antibody (1:1000 dilution; A1253, Abclonal) and GAPDH antibody (1:10000 dilution; 60004-1-Ig, Proteintech).

Techniques: Western Blot, Flow Cytometry, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: Pou3f1 mediates the effect of Nfatc3 on ulcerative colitis-associated colorectal cancer by regulating inflammation

doi: 10.1186/s11658-022-00374-0

Figure Lengend Snippet: Pou3f1 knockdown affected cell proliferation and death in colon tumors of UC-CRC mice. (A-B) Representative immunofluorescent images and quantification results of PCNA (green). DAPI (blue) was used to stain the cell nuclei. (C-D) Representative images and quantification results of TUNEL-positive cells (green). DAPI (blue) was used to stain the cell nuclei. (E) The schematic diagram for the regulatory mechanism of Nfatc3-Pou3f1 in the development of UC-CRC. Data were from n = 6 mice per group. Values were mean ± SD. *, p < 0.05; **, p < 0.01

Article Snippet: Membranes were incubated with primary antibodies, including Nfatc3 antibody (1:1000 dilution; 18222-1-AP, Proteintech), Pou3f1 antibody (1:1000 dilution; A19330, ABclonal), iNOS antibody (1:1000 dilution; A0312, Abclonal), COX-2 antibody (1:1000 dilution; A1253, Abclonal) and GAPDH antibody (1:10000 dilution; 60004-1-Ig, Proteintech).

Techniques: Knockdown, Staining, TUNEL Assay