Fluorescent cells were counted less than a fluorescence microscope, and the numbers were expressed as the percentage of total cellss.d. volume, survival and toxicity were analyzed. AAVP trafficking and TNF- production were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed the combination of AAVP-TNF- and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF- and LCL161 was also significantly more effective than either agent only, showing a synergistic effect without systemic toxicity. by analysis of body mass, feeding status and mobility. All mice were weighed once per week. Analysis of drug combined effects Drug synergy was analyzed and quantified from the drug combination-index (CI) methods using CalcuSyn software (Biosoft, Ferguson, MO, USA).33 The CI method is a mathematical and quantitative representation of a two-drug pharmacologic interaction.33 We used the drug dose for AAVP-TNF- and LCL161 from our tumor growth inhibition experiments and, using the CalcuSyn software, we generated CI values over a range of fraction levels (Fa) from 0.05 to 0.90 (5C90% growth inhibition). A CI of 1 1 shows an additive effect between AAVP-TNF- and LCL161, whereas a CI of <1 shows the presence of synergistic activity. The AAVP trafficking detection by immmunofluorescence assay (IF) with anti-filamentous single-stranded DNA bacteriophage For detection of AAVP, 5??-solid paraffin sections from your resected tumor tissues and normal tissues (liver, kidney, heart, spleen and skeletal muscle) were stained by dual IF.19, 20 The sections were incubated overnight at 4?C in a 1:1000 dilution of rabbit anti-filamentous single-stranded DNA bacteriophage antibody (Sigma Chemical Organization, St Louis, MO, USA) and a concentration of 10?ng?l?1 of antigen affinity-purified rat anti-mouse CD31 antibody (BD Biosciences, San Jose, CA, USA).19, 20 Slides were next incubated with the secondary antibodies (1:200 dilutions each of goat anti-rabbit Alexa Fluor 647 and goat anti-rat Alexa Fluor 488; Invitrogen, Grand Island, NY, USA) for 45?min Csta in the dark.19, 20 The slides were mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylinodole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images were taken using a fluorescence microscope with video camera. The AAVP-mediated TNF- transcription detection by real-time PCR Human TNF- mRNA was measured by reverse-transcriptase-PCR (RT-PCR) with primer-probe sequences unique to human TNF- inserted into RGD-A-TNF-. Total RNA was extracted from frozen tumor and normal tissues (liver, kidney, heart, spleen and skeletal muscle mass) with RNeasy total RNA kit (Qiagen, Valencia, CA, USA). First-strand complementary DNAs were generated from the total RNA, and quantitative RT-PCR was performed. PCR products were A-582941 measured as fluorescent transmission intensity after standardization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal control. The following sense and antisense primers and probes for human TNF- were used: sense primer: 5-TTCAGCTCTGCATCGTTTTG-3 antisense primer: 5-CTCAGCTTGAGGGTTTGCTACA-3, and Probe 5-FAM-TTCTCTTGGCGTCA GATCATCTTCTCGAAC-TAMARA-3.20 The AAVP-mediated TNF- expression by an enzyme-linked immunosorbent assay (ELISA) Levels of human TNF- were assessed by ELISA.19, 20 Total cell lysates from peripheral blood, frozen tumor tissues and frozen normal A-582941 tissues (liver, kidney, heart, spleen and skeletal muscle) were prepared in lysis buffer.19 The amount of protein was quantified using protein assay reagent (Bio-Rad, Hercules, CA, USA). Total protein (100?g) was assayed for human TNF- by ELISA (Biosource, San Francisco, CA, USA).19, 20 Measurement of apoptotic cells in tumor tissues by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay We assessed the apoptotic status in tumor tissues from control and treated mice at days 7 and 21 by TUNEL assay with an Cell Death Detection Kit (Roche Diagnostic, Indianapolis, IN, USA). The tissue sections were treated with proteinase K (10?g?ml?1) for 20?min. A-582941 The sections were next washed twice with PBS, labeled and stained with the TUNEL reaction combination (label plus enzyme solutions) for 60?min at 37?C and washed twice with PBS. The slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). The apoptotic fluorescent cells were counted under a fluorescent microscope, and the figures were expressed as the percentage of total cellss.d. A negative control without enzyme treatment and a positive control with DNase I treatment were also performed. Measurement of the cIAP1 and cIAP2 mRNA expression by real-time RT-PCR We assessed the mRNA expression levels of cIAP1 and cIAP2 in tumor tissues from control and treated mice groups at days 7 and 21 by real-time RT-PCR. The RT-PCR products were measured as fluorescent signal intensity after standardization with a GAPDH internal control. The following primers for cIAP1 and cIAP2 were used: sense primer for cIAP1:.