Fig 1: Ox-LDL increased ADAM17 and sTIMD4 while decreased mTIMD4 level in macrophages. RAW264.7 cells were treated with ox-LDL at the indicated concentrations for 24 h (A, D–G) or with 80 µg/mL ox-LDL for the indicated times (B) or LPS at 1 µg/mL for 24 h (C). After treatment, total cellular proteins, RNA, and treatment mediums were collected. (A–C) Expression of ADAM17, mTIMD4, IL-6, IL-10, pro-IL-1ß, IL-1ß, caspase-1, and IL-18 in cells and sTIMD4 in medium was detected by Western blotting (n = 3). (D and E) Expression of TIMD4, ADAM17, TNF-a, and IL-6 mRNA was determined by qRT-PCR (n = 3–4). HSP90 was used as a loading control. *p < 0.05; **p < 0.01; ns, not significant.
Fig 2: Ox-LDL/LPS activated ADAM17 to increase cleavage of mTIMD4 in macrophages. RAW264.7 cells treated with ox-LDL (80 µg/mL) and TAPI-1 (1 µM) for 24 h. (A) and (B) mTIMD4 expression was detected by immunofluorescent staining (n = 5). (C–F) The protein expression of mTIMD4, sTIMD4 in medium, TLR-4, p-NF-?B, NF-?B, IL-6, caspase-1, pro-IL-1ß, TGF-ß1, and IL-10 were detected by Western blotting (n = 3). (G–J) The mRNA expression mTIMD4, TLR-4, IL-6, and TGF-ß1 was determined by qRT-PCR (n = 3–4). (K–N) RAW264.7 cells were treated with LPS (1 µg/mL) and TAPI-1 (1 µM) for 24 h, and the protein expression of mTIMD4, sTIMD4 in medium, TLR-4, p-NF-?B, NF-?B, IL-6, caspase-1, pro-IL-1ß, TGF-ß1, and IL-10 were detected by Western blotting (n = 3). (O–P) RAW264.7 cells were transfected with 20 nM control si-NC or si-TIMD4 for 48 h and incubated with ox-LDL (80 µg/mL) and TAPI-1 (1 µM) for 24 h, followed by determination of mTIMD4, TLR-4, p-NF-?B, NF-?B, IL-6, IL-10, TGF-ß1, caspase-1, pro-IL-1ß protein expression by Western blotting (n = 3). HSP90 or GAPDH was used as a loading control. ns, not significant. *p < 0.05; **p < 0.01.
Fig 3: Serum sTIMD4 levels were increased associated with CHD. Serum samples collected from healthy controls, CHD, CCS, or ACS patients were used to determine TIMD4 levels by ELISA, followed by comparison with nonparametric tests between healthy controls and CHD patients (Group 1, A) or CCS and ACS patients (Group 2, B). Each dot presents an individual. The whisker plot presents the mean with min to max, and the beard plot presents an average from smallest to largest. (C) ROC curve of serum sTIMD4 for predicting CHD events. AUC is 0.787. The optimal cut-off is 0.34. The sensitivity is 0.749, and the specificity is 0.667.
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