Hek 293t human epithelial kidney cells

Solasonine was purchased from Yuanye Biotech. (Jinan, China). Prostaglandin E2 (PGE2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Hh pathway antagonists GDC-0449 and JQ1 were obtained from Selleckchemicals (Houston, TX, USA). The tumor necrosis factor α (TNF-α), BAY 11-8072 and H89 were purchased from Beyotime (Suzhou, China). Plasmids used in this study were described as previously reported [28 (link)]. The cells used in this study, including light II cells, NIH-3T3 and C3H/10T1/2 mouse embryo fibroblast cells, HEK-293T human epithelial kidney cells, and LS174T colon cancer cells, were all obtained from the American Type Culture Collection (Manassas, VA, USA), and were routinely cultured according to the manufacturer′s instructions. HEK-293T were transfected with ShhN and GFP plasmids. After transfection of 48 h the ShhN conditioned medium (ShhN CM) and GFP CM were collected as previously described [28 (link)].

The K562 human chronic myelogenous leukemia cell line, KB human epidermoid carcinoma cell line, NIH-3 T3 mouse embryo fibroblast cells, and HEK293T human epithelial kidney cells were purchased from the American Type Culture Collection and cultured according to the manufacturer’s instructions. The Dox selected multidrug tolerant K562/A02 subline was obtained from the Institute of Hematology, Chinese Academy of Medical Sciences (Tianjin, China), which was routinely maintained in medium containing 200 ng/ml of Dox [24 (link)]. The VCR selected multidrug tolerant KB/VCR subline was obtained from Zhongshan University of Medical Sciences (Guangzhou, China) and was routinely maintained in medium containing 200 ng/ml of VCR [25 (link)]. Both resistant cells were authenticated by comparing their fold resistance with that of the parental cells and examining the expression levels of ABC transporters. All experiments using K562/A02, and KB/VCR cells were performed with cells growing in the absence of Dox or VCR for at least 5–7 days to avoid drug associated secondary effects.

The NIH-3T3 and C3H/10T1/2 mouse embryo fibroblast cells, HEK-293T human epithelial kidney cells, and LS174T colon cancer cells were obtained from the American Type Culture Collection (Manassas, VA). All these cells were routinely cultured according to the manufacturer’s instructions.
The variant containing the N-terminal signaling domain of the Shh (Shh) conditioned medium (CM) were prepared as previously described [18 (link)]. Briefly, 293 T cells (5 × 10⁶) were seeded in 10-cm dishes. The plasmid harboring the ShhN were transfected into 293 T cells with Lipofectamine 2000 reagent (Invitrogene; Grand Island, NY). The medium (5 ml) in the cells were replaced with fresh medium with 0.1 % serum 24 h post transfection. After 24, the ShhN CM were collected, and were diluted 100-fold prior to be used for experiments.