Cell culture Human colorectal cancer cell lines HCT116 and HT29, and pancreatic adenocarcinoma cell line Panc 1 were obtained from ATCC The cells were maintained in Dulbeccos modified Eagles medium con taining 1 g l glucose supplemen ted with 10% horse serum penicillin streptomycin and two mM glutamine Cells were plated onto Costar plastic culture wells at a density of 50 000 cells cm2 in serum containing medium. The cultures were kept in 95% air 5% CO2 at 37 C. Right after 24 hours the medium was replaced with serum zero cost medium and also the cells had been cultured for 24 hrs prior to stimulation with agonists. Measurement of DNA synthesis Neurotensin, TPA, and inhibitors of PKC and EGF receptor were added to serum starved HCT116 cells as described while in the figure legends, and thymidine was extra twelve hrs just after stimulation. Serum starved HT29 and Panc one cells had been stimulated for 21 hrs with neurotensin and EGF before thymidine was additional.
The cells had been harvested immediately after 3 hrs pulsing with thymidine, and DNA synthesis was measured since the amount of radioactivity incorporated into DNA as these details previously described Briefly, medium was removed, and cells were washed twice with 0. 9% NaCl. The cellular materials was dissolved with one. 5 ml of 0. five N NaOH for three hours at 37 C, collected, mixed with 1. five ml H2O, and precipitated with 0. 75 ml 50% trichloroa cetic acid The acid precipitable materials was transferred to glass fiber filters and washed twice with five. 0 ml 5% TCA, followed by liquid scintillation counting in the filters in the Packard Tri Carb liquid scintillation counter. Inositol phosphate accumulation Cells were labelled with inositol, 2. five uCi ml for 24 hours in serum no cost medium. Medium was eliminated thirty minutes before agonist stimulation and replaced with Krebs Ringer Hepes buffer pH seven.
four, containing ten mM glucose and 15 mM LiCI. HCT116 cells were stimu lated with neurotensin for 30 minutes, as well as the response was stopped by removing buffer and adding 1 ml ice cold 0. 4 M perchloric acid. Samples have been harvested and neutralized with one. 5 M KOH, 60 mM EDTA, 60 mM Hepes, in the presence of Universal indicator. The neutralized Ruxolitinib supernatants were utilized on columns con taining one ml Dowex AG 1 X8 resin and inositol phosphates were eluted with ten ml 1 M ammonium formate 0. 1 M for mic acid. Immunoblotting Aliquots with thirty 000 cells had been electrophoresed on six 12% polyacrylamide gels. This was followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with antibodies against phospho Akt, total Akt, phospho ERK1 2, total ERK, phospho EGFR, complete EGFR, phospho Shc, and complete Shc, respectively.