Fig 1: IL‐1β upregulates human AMTN gene promoter activities. The transcriptional activities of −211AMTN, −353AMTN, −501AMTN, −769AMTN and −950AMTN, were increased by IL‐1β (1 ng·mL−1, 12 h) in Ca9‐22 cells. The results of transcriptional activities obtained from three separate transfections with constructs, pGL3‐basic and −100AMTN to −950AMTN, were combined, and values are expressed with standard errors. *P < 0.05 and **P < 0.01.
Fig 2: Regulatory elements in the proximal promoter of the human AMTN gene. Upper panel: The schematic diagram of human AMTN gene proximal promoter. Lower panel: The nucleotide sequences of the human AMTN gene promoter encompassing an inverted TATA box, inverted CCAAT box, AP1, C/EBP1, Oct1, C/EBP2, YY1, and SP1 are shown from ‐353 to transcription start site.
Fig 3: Effects of IL‐1β on AMTN mRNA and protein levels in Ca9‐22 cells. (A) Dose–response effects of IL‐1β on AMTN mRNA levels in Ca9‐22 cells treated for 12 h. (B) Ca9‐22 cells were treated with or without IL‐1β (1 ng·mL−1) for 3, 6, 12, and 24 h. AMTN and GAPDH mRNA levels were analyzed by real‐time PCR. The experiments were performed in triplicate for each data point. Quantitative analyses of the data sets are shown with standard errors. Significantly different from control, *P < 0.05 and **P < 0.01. (C) AMTN protein levels in Ca9‐22 cells were analyzed by western blotting using anti‐AMTN, anti‐CK19, and anti‐α‐tubulin antibodies.
Fig 4: Immunohistochemical (IHC) staining of dental follicle and permanent PDL tissues.(A, F) IHC staining for AMTN in the dental follicle and (K, P) permanent PDL tissues. (B, G) IHC staining for CXCL13 in the dental follicle and (L, Q) permanent PDL tissue. (C, H) IHC staining for DMP1 in the dental follicle and (M, R) permanent PDL tissue. (D, I) IHC staining for WIF1 in the dental follicle and (N, S) permanent PDL tissue. (E, J) IHC staining for MMP9 in the dental follicle and (O, T) permanent PDL tissue. (Scale bars: 20 µm in A–E and P–T; 100 µm in F–O.)
Fig 5: ChIP analyses of transcription factors binding to C/EBP1, C/EBP2, and YY1 in the human AMTN gene promoter in Ca9‐22 cells. PCR bands amplified and corresponding to DNA–protein complexes immunoprecipitated with antibodies showed that C/EBPβ and YY1 interacted with a chromatin fragment containing the C/EBP1, C/EBP2, and YY1, which were increased in Ca9‐22 cells following stimulation with IL‐1β for 3, 6, 12, and 24 h.
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