USP5 Deletion Inhibits KD Serum Induced-Human Coronary Artery Endothelial Cell Dysfunction by Regulating the NFATC1/TLR4-Mediated NF-κB Signaling Pathway in Kawasaki Disease

Figures

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  Fig. 1. Expression patterns of NFATC1 in KD patients and KD serum-stimulated HCAECs. (A) RT-qPCR assay was applied to measure the expression level of NFATC1 in serum samples from healthy control participants (HC, n = 29) and KD patients (n = 33). (B) The ROC curve analysis of NFATC1 levels for discriminating patients with KD from healthy control. (C and D) HCAECs were exposed to medium with 15% serum from KD patients or HC controls for 24 h. Then, NFATC1 mRNA level and protein level were detected in stimulated HCAECs using RT-qPCR or western blot. *P < 0.05

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  Fig. 2. Influences of NFATC1 silencing on KD serum-induced HCAEC injury. (A) NFATC1 protein level was determined in HCAECs transfected with si-NC or si-NFATC1 using western blot. (B-H) HCAECs were treated with HC serum, KD serum, KD serum + si-NC, or KD serum + si-NFATC1. (B) Cell viability was assessed using CCK-8 assay. (C) EdU-positive cells were measured using EdU assay. (D) Cell apoptosis rate was analyzed using flow cytometry. (E) Bax and Bcl-2 protein level were determined using western blot. (F) The secretions of IL-1β and TNF-α were measured using ELISA kits. (G) The content of ROS was analyzed with DCFDA-cell ROS detection kit. (H) SOD levels was detected using a special kit. *P < 0.05

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  Fig. 3. USP5 affected NFATC1 expression through deubiquitination. (A) The relationship between USP5 and NFATC1 was analyzed using the ubibrowser database. (B) USP5 protein level was measured in HCAECs transfected with si-NC or si-USP5 using western blot. (C) NFATC1 protein level was examined using western blot in HCAECs transfected with si-NC, si-USP5, or si-USP5 + MG132. (D) USP5 protein level was assessed using western blot in HCAECs transfected with Vector or USP5. (E) Influences of USP5 overexpression on NFATC1 protein stability after CHX treatment was detected in HCAECs using western blot. (F) Analysis of NFATC1 protein and ubiquitination levels by Western blot in HCAECs transfected with Flag-NFATC1 + si-NC or Flag-NFATC1 + si-USP5. (G) The binding association between NFATC1 and USP5 in HCAECs using Co-IP assay. *P < 0.05

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  Fig. 4. USP5/NFATC1 regulated KD serum-induced HCAEC injury. (A) NFATC1 protein level was detected in HCAECs transfected with Vector or NFATC1 using western blot. (B-H) HCAECs were treated with HC serum, KD serum, KD serum + si-NC, KD serum + si-USP5, KD serum + si-USP5 + Vector, or KD serum + si-USP5 + NFATC1. (B and C) Cell viability and EdU-positive cells were assessed using CCK-8 assay and EdU assay. (D) Flow cytometry analysis of cell apoptosis. (E) Western blot analysis of Bax and Bcl-2 protein level. (F) IL-1β and TNF-α secretions were assessed using ELISA kits. (G and H) Special assay kits identified of ROS and SOD products. *P < 0.05

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  Fig. 5. NFATC1 interacted with TLR4 to affect its expression. (A) The interaction between NFATC1 and TLR4 was analyzed by STRING database (a useful tool for studying protein–protein). (B) TLR4 protein level was determined using western blot assay in HCAECs treated with HC serum, KD serum, KD serum + si-NC, KD serum + si-NFATC1. (C) Western blot analysis of TLR4 protein level in HCAECs treated with HC serum, KD serum, KD serum + si-NC, KD serum + si-USP5, KD serum + si-USP5 + Vector, or KD serum + si-USP5 + NFATC1. (D) The targeted binding of NFATC1 to TLR4 was verified using Co-IP assay. *P < 0.05

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  Fig. 6. USP5 deubiquitination stabilizes NFATC1 to regulate TLR4/NF-κB signaling pathway. (A and B) p-P65, P65, p-IκBα, and IκBα protein levels were measured using western blot assay in HCAECs treated with HC serum, KD serum, KD serum + si-NC, KD serum + si-USP5, KD serum + si-USP5 + Vector, or KD serum + si-USP5 + NFATC1. (C) Western blot analysis of TLR4 protein level in HCAECs transfected with Vector or TLR4. (D and E) Western blot analysis of p-P65, P65, p-IκBα, and IκBα protein levels in HCAECs treated with HC serum, KD serum, KD serum + si-NC, KD serum + si-NFATC1, KD serum + si-NFATC1 + Vector, or KD serum + si-NFATC1 + TLR4. *P < 0.05