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. 2015 Feb 4;24(10):2808–2825. doi: 10.1093/hmg/ddv042

Figure 7.

Figure 7.

Effects of LDL and ketoconazole treatment on Nsdhlflx5/Y and NsdhlΔ5/Y GCPs in vitro. GCPs were isolated from individual cerebella at P4, split into 4 culture wells with the indicated treatments and analyzed after 48 h. All samples received 1 µg/ml recombinant SHH throughout the 48 h culture. Values represent the mean ± SEM. (A) The relative expression levels of Gli1 were measured by qPCR in Nsdhlflx5/Y and NsdhlΔ5/Y GCPs treated with LDL (5 μg/ml) and/or ketoconazole (2 μM). The results represent 4 independent samples that were each assayed in triplicate with Gapdh used as the endogenous control and normalized to the untreated Nsdhlflx5/Y sample set equal to 1. (B) Percentage of viable cells, as measured by propidium iodide staining of live GCP cultures in situ. Unstained cells were counted as viable. The results are from 4 Nsdhlflx5/Y and 5 NsdhlΔ5/Y independent cultures. (C) Lysed cell cholesterol concentration (μg/mg protein) for aliquots of the cultured GCPs used for qPCR analysis in (A). None of the pairwise comparisons between Nsdhlflx5/Y and NsdhlΔ5/Y samples in the four culture conditions reached statistical significance (P < 0.05) (see text). (D) Lysed cell total methylsterol concentration for the samples used in (C). The concentration of methylsterols was significantly higher in NsdhlΔ5/Y GCPs than Nsdhlflx5/Y GCPs (P < 0.002) in all four culture conditions. (E) Lysed cell total desmosterol concentration for samples used in (A). Desmosterol levels were significantly lower in NsdhlΔ5/Y versus Nsdhlflx5/Y GCPs in both untreated and LDL-treated cultures. Ketoconazole treatment reduced the desmosterol concentration in Nsdhlflx5/Y cells to a level equivalent to that of NsdhlΔ5/Y cells, while addition of LDL to the ketoconazole-treated cultures had no effect on desmosterol.