NCI-H295R–derived conditioned medium inhibits SHH signaling activity. (A) NCI-H295R cells secrete lower amount of endogenously produced SHH than SHH-transfected HEK-293 and HeLa cells. Western blot of SHH-containing conditioned media from NCI-H295R (NCI-Shh-Lpp), HEK-293 (HEK-ShhNc), and HeLa cells (HeLa-ShhNc). Loading volumes and dilutions of the media were adjusted so that all samples have similar blotting intensity. SHH St is control SHH protein, engineered from mouse Shh sequence with an N terminus His tag, enterokinase cleavage sequence (DDDDK), and 2× Ile replacing the first Met (molecular weight = 24.5 kD). The amount of secreted NCI-Shh-Lpp is ∼10× lower than HEK-ShhNc and 60× lower than HeLa-ShhNc. (B) Shh-LIGHT2 cells were treated with the same amount of concentrated or unconcentrated NCI-H295R–conditioned medium, alone or together with HEK-ShhNc. Treatment with the SMO-agonist SAG is a positive control for Shh-LIGHT2 assay activity. Treatment with anti-SHH 5E1 antibody together with HEK-ShhNc is a positive control for the inhibition of HEK-ShhNc signaling activity. (C)Gli1 expression in Shh-LIGHT2 cells treated with 200 nM SAG, 10 ng HEK-ShhNc alone or together with 10 µg/ml 5E1 or increasing amounts of NCI-H295R–conditioned medium (NCI-Shh-Lpp) or the appropriate medium control (Lpp Control). B-actin was used as a reference. Data are normalized to the values for relative Gli1 expression in HEK-ShhNc–treated cells. (D) Shh-LIGHT2 cells were treated with 10 ng HEK-ShhNc together with increasing amounts of 100× concentrated HeLa (not transfected with SHH) or NCI-H295R–conditioned medium. (B–D) Data are presented as mean ± SD, n = 6–9 replicates, pooled from three experiments. **, P < 0.01; ***, P < 0.001; **** P < 0.0001.