Human adrenocortical carcinoma cells do not respond to autocrine noncanonical SHH signaling. (A and B) Measurement of extracellular acidification rate (ECAR; A) and oxygen consumption rate (OCR; B) with Seahorse technology after treatment of NCI-H295R cells grown in serum-free conditions with the SHH pathway inhibitors, 10 µg/ml 5E1 or 10 µM cyclopamine or 200 nM SAG and appropriate controls. n = 12 replicates from two independent experiments, presented as mean ± SD. (C and D) Measurement of intracellular cAMP levels in NCI-H295R cells, cultured in serum-free medium or with the respective treatments (10 µg/ml 5E1, 10 µM cyclopamine, 200 nM SAG and appropriate controls) after 5 min (C) or 24 h (D). Treatment with 20 µM forskolin was used as a positive control for induction of cAMP production in NCI-H295R cells. Data are presented as mean ± SD, n = 6 replicates, pooled from three experiments. 2-DG, 2-deoxy-D-glucose; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; LPP, humapn lipoproteins; mpH, milli pH; Rot/Ant, Rotenone/Antimycin A.