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. 2019 May 27;8(5):509. doi: 10.3390/cells8050509

Figure 3.

Figure 3

Inhibition of ligand-induced Hedgehog signaling. NIH3T3 cells were pretreated for 2 h with the oxysterols or various inhibitors, as indicated, in DMEM containing 5% FBS. Next, cells were treated with CAPAN-1 conditioned medium (CM) in the absence or presence of oxysterols or inhibitors. After 72 h, RNA was extracted and analyzed by Q-RT-PCR for the expression of Hh target genes Gli1 (a,b,d) and Ptch1 (c), and normalized to Oaz1 expression. (e) NIH3T3 cells cultured in 24-well plates were transfected with a Gli response-element reporter (pGL3b-8xGliBS-Luciferase) plasmid and a pTK-Renilla-Luciferase plasmid. 24 h after transfection, cells were treated with the test agents, as indicated for 72 h. Luciferase activity was measured and normalized to the Renilla luciferase activity. (f) NIH3T3 cells were treated with the oxysterols, as indicated, in DMEM containing 5% FBS for 72 h. Q-RT-PCR was performed as described in (a). Data from a representative experiment are reported as the mean of triplicate determinations ± SD (** p < 0.01 vs. CM-treated cells (a–e) or Oxy133 (f); # p < 0.01 vs. control).