Cdna master mix

Cells were harvested for RNA isolation in TRIreagent (Life Technologies, Carlsbad, CA, USA) and stored at −80 °C. RNA was isolated using RiboPure^TMkit (Applied Biosystems, Foster City, CA, USA) and converted to cDNA with high-capacity RNA to cDNA master mix (Applied Biosystems, Foster City, CA, USA). cDNA was diluted 10× and stored at −20 °C until further use. For each gene target 5 µL of cDNA was amplified in duplicate using Fast SYBR Green Mastermix (Applied Biosystems, Foster City, CA, USA) on a StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Primers are listed in Table 1. Mean cycle thresholds were converted to relative expressions by subtraction of the 18S rRNA cycle threshold and determination of 2^−ΔCt [63 (link)].

MDCK cells were grown to about 90% confluence, infected with influenza virus at 0.001 MOI and cultured in the presence of 18-hydroxyferruginol (1), 18-oxoferruginol (2), and oseltamivir. The infected/untreated cells were cultured in the presence of 0.5% DMSO. The medium was removed after 3, 6, 12, and 18 h. Cells were scraped off, washed twice with phosphate buffer saline (PBS), and collected by centrifugation (500× g for 3 min). The total RNA was isolated using the Qiagen RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The specific primers were used for the reverse transcription of viral RNA (vRNA; M gene, 5′-CTTCTAACCGAGGTCGAAACGTA-3′ and 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; NP gene, 5′-AGRTAYTGGGCYATAAGRAC-3′ and 5′-CTTATTTCTTCGGAGACAATGC-3′). GAPDH was used as the internal control for cellular RNAs, with primer sequences of 5′-TCAACGGATTTGGCCGTATTGG-3′ and 5′-TGAAGGGGTCATTGATGGCG-3′. cDNA was synthesized from total RNA using the cDNA Master Mix (Applied Biosystems, CA, USA). qRT-PCR was conducted using 2 μL of cDNA and the Power SYBR Green PCR 2 X Master Mix (Applied Biosystems, CA, USA). qRT-PCR was conducted using the Step One Plus Real-time PCR System, and the data were analyzed with the StepOne software v2.1 (Applied Biosystems, CA, USA).

From rat skeletal muscle, total RNA was extracted from the EDL and Soleus using the RiboPure kit (Applied Biosystems, Cat#AM1924, Foster City, CA, USA) according to the manufacturer’s instructions. RNA concentrations were determined in duplo by spectroscopy (ND-1000 spectrophotometer; Nanodrop Technologies, Wilmington, DE, USA). RNA purity was ensured by 260/280 ratio (range 2.00–2.11, mean 2.04). The muscle total RNA concentration was calculated on the basis of total RNA yield (μg) per weight (mg) of the analyzed sample.
For C2C12 cells, after washing cells with phosphate-buffered saline (PBS), cells were lysed in TRI reagent (Invitrogen, Cat#11312940, Carlsbad, CA, USA) and stored at −80 °C. RNA was isolated using RiboPure kit and converted to cDNA with high-capacity RNA to cDNA master mix (Applied Biosystems, Cat#4388950, Foster City, CA, USA). cDNA was diluted 10 times and stored at −20 °C.