Fig 1: Mechanism diagram: Inhibition of SPI1 by ADAP regulates S100A8/A9 signaling in macrophages to control the development of colitis. In inflammatory macrophages, ADAP upregulates FBXW7 expression and promotes ubiquitin-proteasome-mediated degradation of the transcription factor SPI1, thereby suppressing the expression of downstream effectors S100A8 and S100A9. ADAP deficiency disrupts this checkpoint, leading to SPI1 accumulation and aberrant overproduction of S100A8/A9, which triggers excessive inflammatory responses and exacerbates DSS-induced colitis
Fig 2: ADAP deficiency in macrophages drives NF-κB-dependent transcriptional activation of S100A8/A9. A Western blot analysis of S100A8/A9 and NF-κB pathway-associated proteins in colonic tissues from DSS-treated WT and Adap−/− mice. B RT-qPCR analysis of S100a8/a9 mRNA levels in colonic tissues from DSS-treated WT and Adap−/− mice. C ELISA analysis of S100A8/A9 protein levels in colonic tissues from DSS-treated WT and Adap−/− mice. D RT-qPCR analysis of S100a8/a9 mRNA in WT and AdapKD RAW264.7 cells following LPS stimulation (1 µg/mL, 24 h). E Western blot analysis of S100A8/A9 and NF-κB-related proteins in WT and AdapKD RAW264.7 cells after LPS stimulation (1 µg/mL, 24 h). Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01
Fig 3: ADAP regulates inflammation via the transcription factor SPI1. A Prediction of SPI1 binding sites on the S100A8/A9 promoter. B Chromatin immunoprecipitation combined with CUT&RUN assay showing SPI1 binding to the S100A8/A9 promoter. C-D RT-qPCR and Western blot analysis of SPI1 expression in the colon from WT and Adap−/− mice with DSS treatment. E-F RT-qPCR and Western blot analysis of SPI1 expression in RAW264.7 cells with LPS stimulation (1 µg/mL, 24 h) in WT and AdapKD RAW264.7 cells. G Luciferase reporter assay assessing S100A8/A9 promoter activity in WT and AdapKD RAW264.7 cells after SPI1 knockdown and LPS stimulation (1 µg/mL, 24 h). H Western blot analysis of S100A8/A9 and NF-κB pathway proteins in RAW264.7 cells following SPI1 knockdown and stimulation with LPS (1 µg/mL, 24 h). Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. ns, not significant, *p < 0.05, **p < 0.01
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