Cells were infected with either recombinant MeV Edmonston (recMeV-Edm) [82] (Vero cells, MOI = 1.0) or influenza A/WSN (MDCK cells, MOI = 0.05), followed by removal of inocula one hour post-infection and addition of JMN3-003 in growth media at 0.1 mM or 1 mM. All MeV infected wells received in addition fusion inhibitory peptide (FIP, Bachem) at 100 mM to prevent premature breakdown of the monolayer through viral CPE in the vehicle control wells prior to RNA extraction. Twenty-four (influenza A/WSN) or forty (recMeV-Edm) hours post-infection, total RNA was prepared from all wells using the QIAcube automated extractor and the RNeasy Mini Kit (Qiagen), and subjected to reverse transcription using Superscript II Reverse Transcriptase (Invitrogen). For RNA samples originating from recMeV-Edm infected cells, antigenomespecific primer 5-GGCTCCCTCTGGTTGT or oligo-dT primer (viral mRNA and GAPDH quantification) were used for cDNA priming. In the case of samples originating from influenza A/WSN infected cells, primers for cDNA synthesis were 5-AGTAGAAA-CAAGGTAGTTT (antigenome) or oligo-dT (mRNA and canine GAPDH). Real-time reactions were carried out using an Applied Biosystems 7500 Fast real-time PCR system and iQ Fast SYBR Green Supermix with ROX (Bio-Rad). Probes were a fragment at the N/P junction (MeV antigenomic RNA, 5-AACCAGGTCCACACAG and 5-GTTG TCTGATATTTCTGAC), a fragment of MeV F mRNA (5-GTCCACCATGGGTCTCAAGGTGAACGTCTC and 5-CAGTTATTGAGGAGAGTT), a fragment of human GAPDH (SABiosciences proprietary primers), a fragment of influenza A/WSN segment seven (influenza A/WSN antigenomic RNA, 5-tagctccagtgctggtct and 5-AAGGCCCTCCTTTCAGTCC), and a fragment of canine GAPDH (Qiagen proprietary primer). Melting curves were generated at the end of each reaction to verify amplification of a single product. To calculate DDC T values, CT values obtained for each sample were normalized for GAPDH as reference and then DC T values of JMN3-003-treated samples normalized for the FIP-treated controls. Final quantification was based on three independent experiments in which each treatment condition and RT primer setting were assessed in triplicate. To assess the relative quantities of cellular mRNA, 9610 5 HeLa cells were incubated in the presence of JMN3-003 (0.01, 0.1, 1.0, 10.0 mM final concentration), AS-136A (25 mM), Actinomycine D (5 mg/ml), or vehicle only for six hours at 37uC, followed by preparation of total RNA as described above. Quantitative TaqMan RT-PCR was again achieved using the TaqMan Fast Master Mix (Applied Biosystems) combined with proprietary primer and probe sets specific for Induced myeloid leukemia cell differentiation protein 1-(MCL1), MAPK phosphatase 1 (MKP1), and ankyrin repeat and SOCS boxcontaining protein 7-(ASB7) encoding mRNAs (Applied Biosystems). Samples were standardized for GAPDH as before and normalized values expressed relative to the equally analyzed vehicle-treated controls.