FIGURE 2.

XN decreases production of mature SREBPs. A, Huh-7 cells were depleted of sterols by incubating in medium C for 16 h. The cells were then switched to medium C in the presence of vehicle, 1 μg/ml 25-HC, 10 μm XN, or 30 μm XN. After incubation for 3 h, whole cell extracts underwent immunoblotting (IB) with anti-SREBP-1 (2A4), anti-SREBP-2, or anti-β-actin antibodies. B–E, structures of XN (B), IXN (C), NG (D), and 8-PN (E). F, Huh-7 cells were depleted of sterols by incubating in medium C for 16 h. The cells were then switched to medium C in the presence of 30 μm XN, IXN, NG, or 8-PN. After incubation for 3 h, whole cell extracts were subjected to immunoblotting with the antibodies listed in A. G, CHO-7 and SRD-15 cells were depleted of sterols by incubating in medium F for 16 h. The cells were then switched to medium F in the presence of vehicle, 1 μg/ml 25-HC, or 30 μm XN. After incubation for 3 h, whole cell extracts underwent immunoblotting with the antibodies listed in A. H and I, Huh-7 cells were transfected with either control (siCon) or Insig-1 and -2 siRNA oligonucleotides (siInsigs), cultured with medium A for 24 h, then cultured with medium C for 16 h, and re-fed the medium containing 1 μg/ml 25-HC or 30 μm XN for 3 h before harvest. H, real time PCR analysis was performed, and relative mRNA levels were obtained by normalization to GAPDH mRNA. I, whole cell extracts underwent immunoblotting with anti-SREBP-1 (2A4), anti-SREBP-2, or anti-β-actin antibodies. The same results were obtained in more than three separate experiments.