Figure 4. Lipoprotein-associated SHH... | Rockefeller University Press

$Lipoprotein-associated SHH secreted from adrenocortical carcinoma cells is signaling-inactive, due to an inhibitory molecule(s) also secreted from these cells.(A) Schematic representation of the experiment. Shh-LIGHT2 mouse fibroblasts were treated with conditioned medium containing secreted, lipoprotein-associated SHH. (B) Shh-LIGHT2 cells were treated with unconcentrated NCI-H295R–conditioned medium or with the corresponding amounts of conditioned medium from SHH-transfected HEK-293 and HeLa cells (HEK-ShhNc and HeLa-ShhNc, respectively). Treatment with the SMO agonist SAG is a positive control for the Shh-LIGHT2 assay activity. (C) Shh-LIGHT2 cells were treated with increasing amounts of concentrated conditioned medium from SHH-transfected HEK-293 and HeLa cells (HEK-ShhNc and HeLa-ShhNc, respectively), and from NCI-H295R cells (NCI-Shh-Lpp). (D and E) Shh-LIGHT2 cells were treated with (D) 10 ng HEK-ShhNc or (E) 200 nM SAG, together with increasing amounts of NCI-H295R–conditioned medium. (F–K) Shh-LIGHT2 cells were treated with 10 ng HEK-ShhNc together with increasing amounts of (F) adrenal gland homogenate or PBS lysis buffer control; (G) conditioned medium from primary adrenal gland cell cultures or medium control; (H) NCI-H295R–conditioned medium or Lpp control medium cleared through 10-, 30-, and 100-kD size filters; (I) whole NCI-H295R–conditioned medium (NCI-Shh-Lpp) or size fractions separated by gel filtration chromatography; (J) conditioned medium from NCI-H295R cells treated with 10 µM ketoconazole or vehicle control; (K) endocannabinoid lipids or vehicle control. The Lpp Control represents medium with added human serum lipoproteins, not cultured with NCI-H295R cells. Data are presented as mean ± SD, n = 4–6 replicates, pooled from two or three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 2AG, 2-acylglycerol; NADopa, N-acyldopamine.$

Figure 4.

Lipoprotein-associated SHH secreted from adrenocortical carcinoma cells is signaling-inactive, due to an inhibitory molecule(s) also secreted from these cells. (A) Schematic representation of the experiment. Shh-LIGHT2 mouse fibroblasts were treated with conditioned medium containing secreted, lipoprotein-associated SHH. (B) Shh-LIGHT2 cells were treated with unconcentrated NCI-H295R–conditioned medium or with the corresponding amounts of conditioned medium from SHH-transfected HEK-293 and HeLa cells (HEK-ShhNc and HeLa-ShhNc, respectively). Treatment with the SMO agonist SAG is a positive control for the Shh-LIGHT2 assay activity. (C) Shh-LIGHT2 cells were treated with increasing amounts of concentrated conditioned medium from SHH-transfected HEK-293 and HeLa cells (HEK-ShhNc and HeLa-ShhNc, respectively), and from NCI-H295R cells (NCI-Shh-Lpp). (D and E) Shh-LIGHT2 cells were treated with (D) 10 ng HEK-ShhNc or (E) 200 nM SAG, together with increasing amounts of NCI-H295R–conditioned medium. (F–K) Shh-LIGHT2 cells were treated with 10 ng HEK-ShhNc together with increasing amounts of (F) adrenal gland homogenate or PBS lysis buffer control; (G) conditioned medium from primary adrenal gland cell cultures or medium control; (H) NCI-H295R–conditioned medium or Lpp control medium cleared through 10-, 30-, and 100-kD size filters; (I) whole NCI-H295R–conditioned medium (NCI-Shh-Lpp) or size fractions separated by gel filtration chromatography; (J) conditioned medium from NCI-H295R cells treated with 10 µM ketoconazole or vehicle control; (K) endocannabinoid lipids or vehicle control. The Lpp Control represents medium with added human serum lipoproteins, not cultured with NCI-H295R cells. Data are presented as mean ± SD, n = 4–6 replicates, pooled from two or three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 2AG, 2-acylglycerol; NADopa, N-acyldopamine.

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