Adrenocortical carcinoma cells cultured with serum supplement endogenously produce and secrete SHH, which cofractionates with LDL and HDL. (A) NCI-H295R cells produce and secrete SHH into the culture medium in the presence of serum supplement. (B) Western blot of cell lysates and conditioned medium from NCI-H295R cells, cultured in serum-free conditions or with bovine, mouse, or human serum, probed for SHH. Equal amounts of cell lysates and corresponding conditioned medium were loaded. ACTIN was used as a loading control for the cell lysates. (C) Western blot of culture medium supplemented with different sera, probed for SHH. Conditioned medium from NCI-H295R cells cultured with human lipoproteins (NCI-Shh-Lpp) was loaded as reference. (D–G) Western blots of density gradient fractions of conditioned medium from NCI-H295R cells cultured in serum-free medium (D) or with bovine (E), mouse (F), or human (G) serum, probed for SHH, APOA1, or APOE. Density of lipoprotein classes is according to Jonas and Phillips (2008).