Ls174t colon cancer 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 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.

The predicted LogS and LogD at different pH values for each structure were calculated using tools available at Chemicalize.com. The PDE-5 inhibitory activity of sildenafil and malonyl-sildenafil was measured in vitro using a commercially available kit according to the manufacturer instructions (BPS Bioscience, San Diego, CA, USA). The assay was reproduced in three independent experiments with triplicate wells in each replicate. IC₅₀ values were calculated using curve fitting functions in GraphPad Prism 9 (San Diego, CA, USA).
The cell-based permeability assays were performed using LS-174T colon cancer cells obtained from the American Type Culture Collection. Cells were treated with 1 µM linaclotide together with different PDE5i concentrations for 4 h, followed by processing of cell extracts for cellular cGMP measurement using cGMP ELISA kits according to the manufacturer instructions (Cayman Chemical, Ann Arbor, MI, USA). The calculated EC₅₀ values are the concentration of compound required to increase cellular cGMP levels by 50% of maximum based on predictions determined by curve fitting in GraphPad Prism 9 (San Diego, CA, USA). To assess activation of cGMP signaling by immunoblotting, we used LS174T cells with inducible PKG2 expression that were described recently [41 (link)].