Fig. 2

HDL-bound S1P regulates the transcription of HDRACA in the endothelial cells by affecting the ubiquitination of KLF5. a RT-qPCR showed the effect of W146 on HDRACA levels in HUVECs treated with nHDL or dHDL. b The S1P content in nHDL (n = 20) and dHDL (n = 20). c The levels of HDRACA in HUVECs treated with increasing concentrations of S1P (from 0.5 μM to 8 μM) were assayed by RT-qPCR. d The levels of HDRACA in HUVECs treated with S1P (1 μM), r-ApoM (1 μM), and r-ApoM-S1P (1 μM) were assayed by RT-qPCR. e The middle tracks display the online dataset of KLF5 ChIP-seq in GM12878 (blue; GSE127670), H3K4me3 ChIP-seq in HUVECs (red; GSM945181, GSM733673, GSE96250) and H3K27ac ChIP-seq in HUVECs (yellow; GSM733691). The purple arrow indicates the putative KLF5 binding site in HDRACA promoter based on the conserved KLF5 binding motif (shown on the top) and peaks. f The mRNA levels of KLF5 in HUVECs treated with nHDL or dHDL were assayed by RT-qPCR. g Immunoblot of KLF5 in HUVECs treated with nHDL or dHDL. The representative plots (left) and quantification (right) are shown. h KLF5 ubiquitination in HUVECs treated with nHDL or dHDL was assayed by immunoprecipitation (IP) and immunoblotting (IB). MG132 was added to inhibit KLF5 degradation. i RT-qPCR confirmed the mRNA levels of HDRACA in HUVECs transfected with negative control siRNA or KLF5-siRNAs. j ChIP-RT-qPCR analysis (right) for KLF5 binding to the HDRACA promoter in HUVECs. Normalized data are shown as percentages of the input controls. KLF5 binding to BECN1 promoter served as a positive control. The whole-cell lysate (INPUT) and immunoprecipitated proteins (IP) of each immunoprecipitation were analyzed by immunoblotting (left) for KLF5 or IgG. k Luciferase reporter assays for HUVECs with endogenous KLF5 expression transfected with pGL4.1 reporter plasmids containing deletion HDRACA promoter constructs. HUVECs transfected with a blank pGL4.1 plasmid served as a negative control. l Luciferase reporter assays for HUVECs with endogenous KLF5 expression transfected with pGL4.1 reporter plasmids containing wild type or mutated HDRACA promoters. HUVECs transfected with a blank pGL4.1 plasmid served as a negative control. Data are presented as the mean ± SD. For all the experiments, n = 6. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant