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4), 250 mM NaCl, 0.5% Triton X-100, 50 mM NaF, 2 mM EDTA and 1 mM Na3VO4 on ice for 30 min. Anti-Flag, anti-ERK1/2, anti-phospho-ERK1/2 and anti-cyclinD1 antibodies were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA). Tumor, liver, spleen, lung, kidney tissues were homogenized in lysis buffer on ice for 30 min. Bacterial lysates were prepared from S. typhimurium strain VNP20009 carrying appropriate vectors as described above. Intensity was quantitatively analyzed by Image J software (NIH, Bethesda, MD, USA). Flow cytometric analysis Cells were harvested by trypsinization for 48 h after transfection, part of the cells were labeled with FITC-conjugated-annexin V and PI (BD PharMingen, SanDiego, CA, USA) according to the manufacturer��s instructions and analyzed by flow cytometry for apoptosis. The rest cells were fixed with 70% ethanol overnight. The fixed cells were rehydrated in PBS and subjected to Capmatinib PI/RNase staining followed by fluorescence activated cell sorter scan (FACS) analysis (Becton Dickinson, Mountain View, CA, USA). Data were analyzed using FlowJo analysis software (Tree Star, Ashland, OR, USA). Animal experiments Six-to-seven-week-old female C57BL/6 mice, purchased from the Laboratory Animal Center, Yangzhou University (Yangzhou, China), were housed in environmentally controlled conditions. The study protocol was approved by local institution review boards this website and the animal study was carried out in accordance the ethical guidelines for animal use and care established by Nanjing University (Nanjing, China). The C57BL/6 mice were inoculated Quinapyramine subcutaneously into the mid-right flank with 5 �� 105 B16F10 cells in 0.1 ml PBS. VNP harboring appropriate plasmids were cultured and prepared as described and then injected intraperitoneally with 0.1 ml PBS containing 1 �� 105 colony-forming units (cfu) bacteria into the tumor-bearing mice 7 days post-inoculation. Tumor volume was determined using the formula: tumor volume = length �� width2 �� 0.5. Statistical analysis Data were expressed as the mean �� standard deviation (SD) and data analysis was performed using Graph Pad Prism version 5.0 (Graph Pad Software, San Diego, CA, USA). Paired Student��s t-test analysis was conducted to assess statistical significance. P