Usual epithelial cells were grown in SFM medium underneath same conditions as the carcinoma cells. which was labelled by chemoluminescence, Twenty microgram total RNA from tumor cells and standard epithe lial cells have been separated on 1% agarose gel. After transfer of the RNA onto nylon membrane both hybridization and detection procedures have been carried out in accordance to the producers guidelines. Isolation of PTX PTX was isolated chromatographically through the marine Cnidaria Palythoa caribaeorum and purified as we described earlier, Purified PTX was lyophilized and stored at 20 C. Cytotoxicity assay Quantification of cell death and cell lysis was depending on the measurement of LDH activity launched through the cytosol of broken cells to the supernatant applying a non radioactive LDH detection kit, Cells grown to a monolayer had been incubated for 24 h from the presence or absence of PTX.
Just after centrifugation at 250xg for 10 min. the cell absolutely free culture supernatants have been collected from PTX taken care of and untreated cells and incubated description in accordance to your makers instruction. To calculate % cytoto xicity suitable controls were measured in just about every experiment. Absorbance was measured at 492 nm and 620 nm working with an ELISA reader, Clonogenic assay At day 0, HNSCC cells and normal epithelial cells have been plated in duplicate in six very well plates. One week later on, just after cells had reached confluency, they had been incubated for 24h at a variety of PTX concentrations, Subsequently, cells were washed with PBS, fixed in ethanol and stained with crystal violet, Stained cells were measured by microscopic counting randomly deciding on at least ten middle power magnification fields.
Suggest values and conventional deviation were calculated. JNK3 inhibitory assay Pyrazolourea, LY2940680 a selective inhibitor of JNK3 was obtained from Merck Calbiochem, Germany. Ordinary epithelial cells have been seeded in six well plates and cultured until confluent. The cells had been incubated with pyrazolourea at concentrations ranging from 20 nM to one hundred nM for 3 hours to inhibit the JNK3 protein kinase. Subsequently, cells had been exposed to six ng ml PTX for 24 hrs. Lastly, cell survival was determined employing the crystal violet assay. Animal experiments SCID bg bg mice were obtained from Charles River aged ten to 12 weeks, To the carcino genicity experiments a group of tumor free mice was treated by subcutaneous injection of 0. 5ng PTX within a volume of twenty ul PBS day for five days. Subsequently,the animals were observed over a period of 8 months.