Fig 1: TRIM59 overexpression alleviated the inflammation and apoptosis of the ox-LDL-induced HUVECs via AnxA2. (A) The levels of TNF-a, IL-6, and IL-8 in transfected ox-LDL-induced HUVECs were detected using ELISA. ***, P<0.001 vs. Control, ###, P<0.001 vs. ox-LDL group, $$$, P<0.01 vs. ox-LDL + Ov-TRIM59 + siRNA-NC. (B) The apoptosis of the transfected ox-LDL-induced HUVECs was detected using TUNEL. DAPI was used for staining. ***, P<0.001 vs. Control, ###, P<0.001 vs. ox-LDL group, $$$, P<0.01 vs. ox-LDL + Ov-TRIM59 + siRNA-NC. (C) The expression levels of Bcl-2, Bax, and cleaved caspase3 in the transfected ox-LDL-induced HUVECs were detected using western blot. ***, P<0.001 vs. Control, ###, P<0.001 vs. ox-LDL group, $$, P<0.01 and $$$, P<0.01 vs. ox-LDL + Ov-TRIM59 + siRNA-NC. TRIM59, tripartite motif-containing protein 59; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; Ov, overexpression; NC, negative control; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal-deoxynucleotidyl transferase mediated nick end labeling; DAPI, 4',6-diamidino-2-phenylindole.
Fig 2: TRIM59 could bind to AnxA2 and promote AnxA2 expression in the HUVECs. (A) The mRNA and protein expression levels of AnxA2 in the ox-LDL-induced HUVECs were detected using RT-qPCR and western blot. **, P<0.01 and ***, P<0.001 vs. Control. (B,C) The binding of TRIM59 and AnxA2 was detected using Co-IP. ***, P<0.001 vs. Anti-TRIM59 group vs. Anti-AnxA2 group, #, P<0.05, ##, P<0.01 and ###, P<0.001 vs. IgG group. (D) The mRNA and protein expression levels of AnxA2 in the TRIM59-overexpressed ox-LDL-induced HUVECs were detected by RT-qPCR and western blot. ***, P<0.001 vs. Control, ###, P<0.001 vs. ox-LDL + Ov-NC group. TRIM59, tripartite motif-containing protein 59; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Ov, overexpression; NC, negative control; AnxA2, annexin 2; Co-IP, co-immunoprecipitation.
Fig 3: TRIM59 overexpression alleviated the viability damage of the ox-LDL-induced HUVECs via AnxA2. (A) The transfection efficacy of siRNA-AnxA2 was detected using RT-qPCR and western blot. ***, P<0.001 vs. siRNA-NC, ###, P<0.001 vs. siRNA-NC. (B) The viability of the transfected ox-LDL-induced HUVECs was detected using CCK-8 assay. (C) The levels of LDH in transfected ox-LDL-induced HUVECs was detected using LDH assays. ***, P<0.001 vs. Control, ##, P<0.01 and ###, P<0.001 vs. ox-LDL group, $$, P<0.01 vs. ox-LDL + Ov-TRIM59 + siRNA-NC group. TRIM59, tripartite motif-containing protein 59; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; LDH, lactate dehydrogenase; Ov, overexpression; NC, negative control; AnxA2, annexin 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, cell counting kit-8.
Fig 4: TRIM59 overexpression suppressed THP-1 adhesion to the ox-LDL-induced HUVECs via AnxA2. (A) The THP-1 adhesion to the ox-LDL-induced HUVECs was detected using monocyte adhesion assays. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF AM) is used for staining. (B) The expression levels of ICAM-1 and VCAM-1 in the transfected ox-LDL-induced HUVECs were detected using western blot. ***, P<0.001 vs. Control, ###, P<0.001 vs. ox-LDL group, $$$, P<0.01 vs. ox-LDL + Ov-TRIM59 + siRNA-NC. TRIM59, tripartite motif-containing protein 59; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; Ov, overexpression; NC, negative control; AnxA2, annexin 2.
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