TMA block sections were subjected to an immunohistochemical analy

TMA block sections were subjected to an immunohistochemical analysis selleck chem inhibitor using anti-CRKL monoclonal antibody (Y243; 1:100 dilution), Histofine Simple Stain Max-Po (Multi), and 3,3′-diaminobenzidine … DNA fluorescence in situ hybridization (FISH) FISH was performed as previously described [16-19]. Tissue slides were hybridized with a Spectrum Orange-labeled BAC clone (RP11-801O20 and RP11-1058B20) for the CRKL locus (Advanced GenoTechs Co., Tsukuba, Japan) and a Spectrum Green-labeled control probe for the near centromere locus on chromosome 22 (BAC clone: RP11-232E17). 4′,6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) was used for nuclear staining (Figure (Figure33). MTT assay and direct cell counting In the experiment involving treatment with the CRKL-targeting peptide, an MTT assay was performed to assess cell viability in Figure Figure4G.

4G. The cells were cultured with the indicated concentration of CRKL-targeting peptide or dimethyl sulfoxide (DMSO) at 37��C for 72h, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO) was then added at a final concentration of 0.25mg/mL. After incubation at 37��C for 4h, absorbance was measured at a wavelength of 570nm using a microplate reader. Cells grown in complete medium with DMSO alone were used as controls. The final concentration of DMSO was set to 0.2%. To assess cell proliferation in Figure Figure4H,4H, the cells were cultured with CRKL targeting peptide or DMSO at 37��C for 72h. Cell proliferation was measured by directly counting the cells using a hemocytometer, as described previously [20].

Figure 4 Responses of the MKN74 gastric cancer cell line withCRKLamplification to treatment with BMS354825 (a dual Src/BCR-ABL kinase inhibitor) and CRKL-targeting peptide. (A) Viability of MKN74 cells treated with BMS354825 but not those treated with AMN107 (a … QRT-PCR Total RNA was extracted using Isogen (Nippongene, Tokyo, Japan) or an RNeasy Plus mini kit (Qiagen, Valencia, CA) and converted to cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-time QRT-PCR was performed using the cDNA and Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) on a StepOne Real-Time PCR system (Applied Biosystems).

The following PCR primers were used: 5′-CAA CCT GCC TAC AGC AGA AGA TAA-3′ and 5′-CGG CAT CAT TCC CAG GAA-3′ for the CRKL transcript, Entinostat and 5′-GGT GGT CTC CTC TGA CTT CAA CA-3′ and 5′-GTT GCT GTA GCC AAA TTC GTT GT-3′ for the transcript of a housekeeping gene, GAPDH. The relative amounts of CRKL transcript were normalized to those of the GAPDH transcript. Western blot analysis Cells were lysed, and the protein concentration was quantified using a BCA protein assay kit (Pierce, Rockford, IL). The proteins were electrophoresed and transferred to a PVDF membrane (GE Healthcare Bio Science, Piscataway, NJ).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>