Clinical follow up data were available for 74 patients, while 128 patients were excluded for lack of information. The study was carried out in selleck chemicals llc accordance with the institu tional ethical guidelines and was approved by the Medical Ethics Committee of Southern Medical Univer sity. Informed consent was obtained from Inhibitors,Modulators,Libraries all patients. Cell culture and treatments SW480 and SW620 cell lines were obtained from Ameri can Type Inhibitors,Modulators,Libraries Culture Collection. SW480CD24 and SW480VEC cells were established, as described in a previous study. Cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1000 ug ml G418 at 37 C with an atmosphere of 5% CO2 and subcultured Inhibitors,Modulators,Libraries by digestion with 0. 05% trypsin. For PP2 treatment, cells were seeded in serum free medium overnight, followed by the addition of PP2 as indicated for 30 min at 37 C.

Medium containing the PP2 inhibitor was renewed after 30 min of incubation. RT PCR Total RNA was isolated from cells using Trizol. Next, 2 ug of RNA sample was subjected to reverse transcription using a RevertAid First Strand cDNA Synthesis Kit. PCR was initiated Inhibitors,Modulators,Libraries by 5 min incubation at 94 C, 36 cycles of denatur ation at 94 C for 45 s, annealing at 56 C and extension at 72 C for 50 s, and ended after a 7 min extension at 72 C using a PCR kit. The experiments were repeated twice and GAPDH mRNA was amplified simultaneously as an internal control. Preparation of siRNA and transfection A siRNA duplex targeting CD24 and a negative control sequence were designed and synthesized by GenePharma Co.BLAST research con firmed the unique sequence of the siRNA.

Lyn siRNA was purchased from Santa Cruz Biotechnology. The specific and negative con trol siRNAs were transfected into SW480CD24 and or SW620 cells 24 h prior to Western blotting analyses or cell invasion assays using lipofectamineTM 2000 reagent according to the manufacturers Inhibitors,Modulators,Libraries instructions. Western blotting analysis Whole cell lysates were prepared as described previously. Equal aliquots of total cell lysates were elec trophoresed on denaturing sodium dodecy1 sulfate polyacrylamide gel electrophoresis gels. The proteins were then transferred to polyvinylidene difluoride membranes. The blots were probed with primary antibodies followed by the horserad ish peroxidase conjugated secondary antibody. Antigen antibody complexes were visualized using an enhanced chemiluminescence system.

GAPDH served as the loading control. Immunofluorescence Immunofluorescent selleck products staining was performed as described. In brief, cells grown on cover glass were fixed in phosphate buffered saline containing 4% parafor maldehyde for 30 min at room temperature. Nonspecific binding was blocked by incubation with 1% bovine serum albumin. Cells were subsequently reacted with an appropriate primary antibody for 1 h, then washed and stained with TR or fluorescein isothiocyanate conjugated second antibodies.