5 uM Sorafenib or Regorafenib was added. Photographs were taken of each well immediately and after 24 h and 48 h. The values were expressed as percentage of migration, with 100% being selleck Ruxolitinib when the wound was completely closed. The results were representative of three independent experiments. Invasion assay Cell invasion assays were performed using Matrigel coated Transwells as previously described. Briefly, 2. 5 uM Sorafenib or Regorafenib treated cells were suspended in low serum medium. Medium containing different hPL or FBS concentrations was added to the bottom wells. After incubation of 24 h, the invading cells were fixed and Inhibitors,Modulators,Libraries stained. The images were acquired and analyzed counting the cells with Image J Software. Values obtained were expressed as fold increase of invading cells, setting the cell counts of control cells as one.
Results were representative of three independent experiments. Apoptosis assays Annexin V The Muse Annexin V Dead Cell Assay Kit for quantitative analysis of live, early Inhibitors,Modulators,Libraries late apoptotic and dead cells Inhibitors,Modulators,Libraries was used with a Muse Cell Analyzer. Briefly, the assay utilizes Annexin V to detect PS on the external membrane of apoptotic cells. A dead cell marker is also used. PLC PRF 5 cell line, including positive and negative controls, were cul tured in 1% FBS medium supplemented with a volume of hPL corresponding to 3. 75 107 platelets ml or with an equivalent percentage of serum for 48 h. The cells were then processed as described in the users guide. Caspase 3 7 quantitative measurements The Muse Caspase 3 7 kit permits simultan eous evaluation of apoptotic status based on Caspase 3 and 7 activation and cellular plasma membrane permeabilization.
The assay provides rela tive percentage of cells that are live, early late apoptotic or dead. Cells were cultured as described above and processed according to the users guide. Western blots We analyzed the MAPK signaling and anti apoptosis markers in Hep3B cells treated with 2. 5 uM Sorafenib or Inhibitors,Modulators,Libraries Regorafenib and hPL by Western blot, as previously de scribed. In brief, cells were washed twice with cold PBS and then lysed in RIPA buffer. After quantization of protein concentration, equal amount Inhibitors,Modulators,Libraries of protein were resolved on SDS PAGE and transferred to polyvinyldifluoride filters. The blots were blocked with 5% nonfat dry milk for 2 h at room temperature and then probed with primary anti body overnight at 4 C. The primary antibodies were directed against the following proteins, ERK and phospho ERK, JNK and phospho Nutlin 3a JNK, p38 and phospho p38, STAT3 and phospho STAT3, AKT and phospho AKT, survivin, Bcl xL, Bax, Bim and B actin. After three washes, incubation was followed by the reaction with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature.