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. 2016 Mar 16;291(20):10747–10758. doi: 10.1074/jbc.M116.722272

FIGURE 2.

FIGURE 2.

NGF increases LDLR expression and leads to SREBP2 cleavage in Huh7 cells. Human Huh7 hepatocytes were stimulated with NGF as indicated. Immunoblotting was done using specific antibodies against LDLR and SREBP2 as described under “Experimental Procedures” using anti-β-actin as control. For statistics, all values are given as mean ± S.E. A, NGF elevated LDLRs in a concentration-dependent (upper panel) and time-dependent manner (lower panel). Left, immunoblot. Right, quantification was done using ImageJ for 50 ng/ml NGF. n = 4. **, p < 0.01 for NGF versus C. B, cells were incubated with 50 ng/ml NGF or with 1 μm simvastatin (Statin) for 24 h. SREBP2 was cleaved as shown by an antibody recognizing the carboxyl-terminal part of the protein. Left, immunoblot. Right, quantification. n = 3. *, p < 0.05 for NGF and for statin versus C. **, p < 0.01 for NGF + statin versus C. C, cells were stimulated with 50 ng/ml NGF for 3–24 h, and nuclear fraction was prepared as described under “Experimental Procedures.” The presence of SREBP2 in the nucleus was analyzed using an antibody against the transcriptionally active amino-terminal part of the protein. Histone H3 was used as a marker for cell nucleus. Typical experiment is shown and was repeated three times with similar results. D, LDLR promoter assay. Cells were transfected with wild-type and mutant LDLR promoter lacking the SREBP2-binding site and treated for 24 h with 50 ng/ml NGF or 1 μm simvastatin (Statin) alone or together. Luciferase activity was measured as described under “Experimental Procedures,” and the ratio of firefly versus Renilla luciferase activity is given. NGF and statin increased the activity of the wild type but not that of the mutant promoter. The effects of NGF and statin were additive. n = 4. *, p < 0.05 for NGF versus C; **, p < 0.01 for statin versus C; and ***, p < 0.001 for NGF + statin versus C. E, LDL uptake. Cells were treated with 50 ng/ml NGF or with 5 ng/ml pro-NGF for 24 h and 5 μg/ml 3,3′-dioctadecylindocarbocyanine-labeled LDL was added for the last 6 h. NGF and pro-NGF increased the uptake of LDL particles. NC is a control in the presence of Dynasore to inhibit endocytosis. **, p < 0.01 for NGF versus C, and for NC versus C. ***, p < 0.001 for pro-NGF versus C.