GBd15 and TGBC2TKB cells were seeded onto 96-well plates at a density of 5000 cells/well and incubated with recombinant human Shh (rhShh; R&D Systems, Minneapolis, MN, USA) or the Smo inhibitor cyclopamine (Toronto Research Chemicals, North York, Canada) for 24, 48, or 72 h. Cell proliferation was determined by the absorbance at 492 nm (ref. 620 nm) using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Forty-eight hours after Smo, MMP-2, and MMP-9 siRNA transfection, the cells were reseeded onto 96-well plates and the proliferation rates were measured.
Rhshh
Manufactured by R&D Systems
Sourced in United States
The RhShh is a laboratory equipment product designed for specific research and development applications. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Cyclopamine, by LGC (1 mentions)
Cell count reagent sf, by Nacalai Tesque (1 mentions)
Mtt stock solution, by Merck Group (1 mentions)
El800 microplate reader, by Agilent Technologies (1 mentions)
Tpc 1, by American Type Culture Collection (1 mentions)
Bcpap, by American Type Culture Collection (1 mentions)
4 protocols using rhshh
1
Evaluating Hedgehog Pathway Regulation
2
Radioiodine Uptake Assay in Cultured Cells
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Cells were cultured in 24-well plates (105 cells/0.5 ml) and treated with or without 0.5 µg/ml rhSHH (R&D Systems, Inc.) for 24 h, subsequently, radioiodine uptake assays were performed. Cells were washed twice with 0.5 ml PBS and subsequently incubated with 0.5 ml of serum-free DMEM/F-12, which contained µCi carrier-free Na131I (0.1 m) (Atomic Hi-tech Radiation Co., Ltd., Beijing, China), with or without perchlorate (100 µM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Perchlorate was used as a NIS-competitive inhibitor for radioiodine uptake. Following 1-h incubation at 37°C, the radioiodine-containing medium was removed and cells were rinsed twice with 1 ml PBS. Subsequently, cells in each well were lysed with NaOH and washed twice with PBS. The cell-associated radioactivity of the collected lysed cells was measured using a gamma counter.
3
Evaluating Shh and Metformin Effects
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The cells were seeded in 96-well plates in complete culture medium, and after 24 h of growth, the cells were exposed to recombinant human Shh (rhShh) (1 µg/ml; R&D Systems, Minneapolis, MN, USA), metformin (3 mM) or a combination of both. Following further incubation at 37°C in a humidified atmosphere with 5% CO2, 50 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/ml; Sigma Chemical) were added to each well at the 0, 12, 24, 48 or 72 h time points, and the plates were incubated for an additional 4 h at 37°C. The solution was then removed from each well, and 150 µl of dimethyl sulfoxide were added. Following gentle agitation, the absorbance at 490 nm was measured using an EL800 microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Experiments were independently performed in triplicate, and 4 parallel samples were measured each time.
4
Shh Signaling in Papillary Thyroid Cancer
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The human Nthy-ori3-1, and PTC cell lines, TPC-1 and BCPAP were obtained from the American Type Culture Collection (Manassas, VA, USA). Cell lines were authenticated by short-tandem repeat profiling performed by BMR Genomics (Padua, Italy). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare) in a 95% humidified atmosphere with 5% CO2 at 37°C. The recombinant human Shh N-terminal peptide (rhSHH; R&D system, Minneapolis, MN, USA) was added to the culture medium. Human PTC specimens and their adjacent normal thyroid tissues (30 pairs) were collected from 30 patients (9 males and 21 females; age 27–63 years) who underwent surgery according to an approved human protocol at the Weifang People's Hospital (Weifang, China), between January 2016 and December 2016. All patient materials were obtained with written informed consent.
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