Measurement of cell viability by MTT The viability of chondrosarcoma cells was measured by methyl thiazolyl tetrazolium assay. Cells had been plated onto 96 nicely plates at a density of 5000 cells per effectively. 6 hours just after transfection with precise siRNA or plasmid, the serum cost-free medium was replaced by com plete medium. The transfection was repeated soon after 48 hrs. MTT reagent in 180 ul medium was extra at 0, 24, 48, 72 and 96 hours and incubated for four hours at 37 C. Subsequent, supernatant was removed and 150 ul dimethyl sulphoxide was additional to each and every properly. After the plate was shaken on a rotary platform for 10 min, extinction at wavelength 490 nm was measured. Measurement of cell proliferation Cell proliferation of chondrosarcoma cells was measured by analyzing BrdU incorpora tion into newly synthesized DNA employing a commercially obtainable ELISA chemiluminescence assay.
Cells were plated out in 96 nicely microtiterplates at a density of 5000 cells per properly and incubated for 24 hours prior the knock down of survivin was performed. 24 following the transfection of specific siRNA the cells have been pulsed for BrdU incorporation in excess of four hrs. ELISA was carried out in accordance further information for the makers instructions. Chemiluminescence values had been measured by an automated luminometer. RNA extraction and true time PCR Survivin mRNA expression was assayed by doing serious time PCR as described in. In quick, RNA was extracted by column purification working with the RNeasy micro kit and RNA transcribed into cDNA. Survivin mRNA expression was detected by a set of intron spanning primer sequences for human survivin and was verified by the application of an independent primer set.
Handle was human b actin. For primer specifics see table 4. All primers have been utilized at a concentration of 300 nmol L and 55 selleck C annealing temperature. A business 2× SYBR Green PCR Combine was used according on the suppliers guidelines. PCR was performed with 50 cycles, taking two ul of cDNA in to the reaction with an finish volume of 25 ul. Values for survivin had been linked to their controls employing the two ct calculation technique. Statistics No less than 3 replicates for every experimental situation were carried out, as well as presented final results were repre sentative of these replicates. All values are presented as implies SEM. College students paired t test was applied to reveal statistical significances. P values less than 0.
05 had been thought of major. Statistical analyses were per formed making use of SPSS Program for Windows. Final results Survivin is expressed in human chondrosarcoma As being a 1st stage, we characterized survivin expression and subcellular distribution in human chondrosarcoma by immunohistochemistry. The staining of paraffin embedded samples revealed striking expression of survi vin protein in all chondrosarcomas analyzed. Higher magnification displays the powerful, predominantly cytoplasmatic subcellular distri bution of survivin protein. In grade III chondrosarcoma, approximately 30% of visi ble nuclei stained good for survivin protein. Impor tantly, cells displaying mitotic structures and tumor giant cells displayed the strongest staining intensity.
To ascertain the specificity with the pattern of staining, we aimed to confirm these findings with several independent antibodies. Altogether, we confirmed the end result with two polyclonal and two monoclonal anti bodies, wherever omission of key antibody gave no sig nal. To strengthen further the evidence of survivin expression in chondrosarcoma we aimed to verify protein expression with techniques aside from immunohistochemistry. Consequently, tissue lysates of three higher grade chondrosarcomas showed specific signals for survivin protein by immuno blotting. To ascertain the correct molecular bodyweight of 16.