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. 2020 Nov 23;296:100032. doi: 10.1074/jbc.RA120.015420

Figure 4.

Figure 4

SENP1 counteracts IDOL-induced degradation of LDLR and augments LDLR-dependent endocytosis of LDL.A, Huh7 cells were transfected as indicated. After 32 h, cells were subjected to cholesterol depletion by incubating with medium A plus 5% lipoprotein-deficient serum, 1 μM lovastatin, and 10 μM mevalonate for 16 h. Cells were then harvested for immunoblotting analysis. B, densitometric analysis of the LDLR bands in (A). Values are normalized to the value obtained from cells transfected with LDLR only and presented as mean ± SEM (n = 3 independent experiments). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, no significance. One-way ANOVA. C, Huh7 cells were transfected as indicated. After 32 h, cells were subjected to cholesterol depletion for 16 h and then harvested for immunoblotting analysis. D–E, CRL1601 cells (WT) and two clones of CRL1601 cells stably expressing SENP1-Flag (SENP1-OE1 and SENP1-OE2) were subjected to cholesterol depletion for 16 h and then harvested for immunoblotting (D) or RT-qPCR analysis (E). Data are normalized to WT cells and presented as mean ± SEM (n = 3 independent experiments). ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significance. One-way ANOVA. F, CRL1601 and SENP1-OE1 cells were depleted of cholesterol at 4 °C for 30 min and then incubated with 10 μg/ml Dil-low-density lipoprotein (LDL) at 4 °C for 1 h. Cells were rinsed with PBS and cultured in cholesterol depletion medium at 37 °C for the indicated time points. Scale bar, 20 μm. G, Quantification of the relative MFI of internalized DiI-LDL in (F). 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 (C) are representative of three independent experiments. Results in (D) and (F) are representative of two independent experiments. IB, immunoblotting; IDOL, inducible degrader of the low-density lipoprotein receptor; LDLR, low-density lipoprotein receptor; MFI, mean fluorescence intensity; ns, no significance; SENP, SUMO-specific peptidase; WT, wild-type.