Response to doxorubicin of MDA/LUC-shUGT8 cells with silenced expression of UGT8 gene and control MDA/LUC cells.

<p>(A) Western blot analysis of anti-caspase-3 mAbs binding to cellular proteins of control MDA/LUC and MDA/LUC-shUGT8 cells with decreased expression of UGT8, grown at 70% confluence in the presence of increasing amounts of doxorubicin (0.005–0.5 µM) for 48 h. Cell lysates, equivalent to 40 µg protein, were separated by SDS-PAGE under reducing conditions on a 13% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control. (B) Survival of control MDALUC cells and MDA/LUC-shUGT8 cultured in the presence of increasing concentrations of doxorubicin (0.001–1.0 µM) for 48 h. Cell viability was determined using MTT assay as described in the “Materials and Methods”. Data represent the mean ± SD of six replicates from two independent experiments. (^*p = 0.0472, two-way ANOVA test).</p

Characteristic of MDA-MB-231/LUC-shUGT8 cells with silenced UGT8 gene expression (A).

<p>Expression of UGT8 and GCS mRNAs in control MDA-MB-231 cells transduced with vector alone (MDA/LUC) and MDA-MB-231 cells tranduced with pLVTHM/LUC-shUGT8 construct (MDA/LUC-shUGT8). UGT8 and GCS levels were normalized against β-actin. (B) Western blot analysis of anti-UGT8 rabbit polyclonal antibodies binding to cellular proteins of control MDA/LUC and sh-transduced MDA/LUC-shUGT8 cells. Cell lysates, equivalent to 40 µg protein, were separated by SDS-PAGE under reducing conditions on a 10% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control. (C) Immunostaining of neutral glycolipids from control MDA/LUC and sh-transduced MDA/LUC-shUGT8 breast cancer cell lines, separated by HP-TLC, with anti-GalCer rabbit polyclonal antibodies. For the analyzed cell lines, an aliquot of total neutral glycolipids corresponding to 1×10⁷ cells were applied to the HP-TLC plate. (D) proliferation of control MDA/LUC and sh-transduced MDA/LUC-shUGT8. Cell proliferation was determined using SRB assay as described in the “Materials and Methods”. The values are shown as the mean ± SD of eight independent replicates.</p

Immunohistochemical analysis of tumor xenografts of MDA/LUC-shUGT8 cells with silenced expression of UGT8 gene and control MDA/LUC cells.

<p>(A) Immunohistochemical staining with monoclonal antibody against Ki67 antigen of tumor sections after subcutaneous implantation of control MDA/LUC cells (I) and sh-transduced MDA/LUC-shUGT8 cells with decreased expression of UGT8 (II) into nu/nu mice. The numbers of Ki67-positive cells in MDA/LUC (I) and sh-transduced MDA/LUC-shUGT8 tumors were compared by Mann-Whitney U-test (^***p<0.0001). (B) TUNEL technique after subcutaneous implantation of MDA-M/LUC cells (I) and MDA/LUC-shUGT8 cells (II) breast cancer cells into nu/nu mice. Original magnification: x100. The numbers of apoptotic cells in MDA/LUC (I) and sh-transduced MDA/LUC-shUGT8 tumors were compared by Mann-Whitney U-test (^*p = 0.0432).</p