Fig 2: 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

Fig 3: TSF alleviated NFATC2 nuclear translocation in AGEs-stimulated primary mice podocytes. (a) Representative immunofluorescence staining and quantitative analysis of NFATC2 (scale bars, 50 µm; 30 cells evaluated per experiment, n = 3 experiments). Nuclei were stained with DAPI. (b) Immunofluorescence staining for NFATC3 (scale bars, 50 µm). The data were expressed as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. BSA group; *P < 0.05, **P < 0.01 vs. AGEs group; ?P < 0.05, ??P < 0.01 vs. AGEs + TSF group. Statistically analyzed via a one-way ANOVA with Dunnett's correction.

Fig 4: Pou3f1 was required for Nfatc3-induced inflammation in LPS-treated macrophages. (A) The relative mRNA levels of IL-1ß, IL-6, TNF-a 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

Fig 5: 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