Figure 5.

SENP1 deficiency reduces LDLR expression and LDL endocytosis.A, Huh7 cells were transfected with scrambled siRNAs (siCtrl) or two individual siRNAs targeting SENP1 (siSENP1-1 and siSENP1-2). After 32 h, cells were depleted of cholesterol for 16 h and harvested for immunoblotting (A) or RT-qPCR analysis (B). Data are normalized to control cells and presented as mean ± SEM (n = 3 independent experiments). ∗p < 0.05, ns, no significance. One-way ANOVA. C, Sanger sequencing analysis of IDOL knockout (KO) cells bearing a frameshift mutation in the exon 1 of the IDOL gene. D, Huh7 and IDOL KO cells were transfected with scrambled siRNAs or two individual siRNAs targeting SENP1. After 32 h, cells were depleted of cholesterol for 16 h and harvested for immunoblotting. E–F, two lines of Huh7 cells lacking SENP1 (SENP1 KO1 and SENP1 KO2) were depleted of cholesterol for 16 h and harvested for immunoblotting (E) or RT-qPCR analysis (F). Data are normalized to control cells and presented as mean ± SEM (n = 3 independent experiments). ns, no significance. One-way ANOVA. G, Huh7 cells and SENP1 KO1 cells were treated as depicted in Figure 4F. Scale bar, 20 μm. H, Quantification of the relative MFI of internalized DiI-LDL in (G). The relative MFI of internalized DiI-LDL in WT cells at 1 h is defined as 1. Data are presented as mean ± SEM (n = 100 cells from 2 independent experiments). ∗∗∗∗p < 0.0001. Unpaired two-tailed Student’s t test. Results in (A) and (D) are representative of three independent experiments. Results in (E) and (G) are representative of two independent experiments. IDOL, inducible degrader of the low-density lipoprotein receptor; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; MFI, mean fluorescence intensity; SENP, SUMO-specific peptidase; WT, wild-type.