Cell-free supernatants were then removed and resolved with 150 μL DMSO. The OD570 nm was measured on a microplate reader. The minimal inhibitory concentration (MIC) of farrerol and other commonly used antibiotics for each isolate was determined using the broth microdilution method with an inoculum of 5 × 105 CFU mL−1 according to the Clinical and Laboratory Standards Institute guidelines, and incubated for 24 h at 37 °C (CLSI, 2005). All tests were performed in duplicate. Bacteria were cultured in MHB at 37 °C, with graded subinhibitory concentrations of farrerol, until the postexponential
growth phase (OD600 nm of 2.5) was reached. Culture supernatants were collected by centrifugation. Total haemolysis http://www.selleckchem.com/products/Roscovitine.html of culture supernatants were evaluated as described previously (Qiu et al., 2010b) using rabbit erythrocytes. Staphylococcus Dasatinib research buy aureus strains ATCC 29213, MRSA 2985 and MRSA 3701 were grown, and supernatants were prepared in the same manner as for
the haemolysis assay. Samples (20 μL) of culture supernatants were boiled in Laemmli sample buffer and loaded on a 12% sodium dodecyl sulphate-polyacrylamide gel (Laemmli, 1970). Western blotting was performed as described by Xiang et al. (2010) and the product instructions for Amersham ECL Western blotting detection reagents (GE Healthcare, UK). Antibody to the α-toxin was obtained from Sigma-Aldrich. A 100-μL volume of supernatant from the postexponential phase (OD600 nm of 2.5) cultures was added to 1 mL of azocasein (Sigma-Aldrich) and incubated at 37 °C for 1 h. After incubation, the reaction was stopped by adding 1 mL of 5% (w/v) trichloroacetic acid, and undigested azocasein was allowed to precipitate for 30 min. The mixture was then centrifuged at 10 000 g for 10 min, and A328 nm of the supernatant was read. Staphylococcus aureus strain ATCC 29213 was incubated with or without the addition of subinhibitory concentrations
of farrerol in the same manner as for the haemolysis assay. Total RNA from the bacterial cultures was extracted as described previously (Qiu et al., 2010a). RNA was reverse transcribed into cDNA using the TaKaRa RNA PCR kit Clomifene (AMV) Ver. 3.0 (Takara, Kyoto, Japan), according to the manufacturer’s instructions. The primer pairs used in real-time RT-PCR are listed in Table 1. The PCR was performed using Sybr green. The reagents consisted of 12.5 μL 2 × SYBR Premix Ex Taq (Takara), 0.5 μL of each primer (10 μM) and 1 μL of sample cDNA in a final volume of 25 μL. The reactions were performed using the 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France). Cycling conditions consisted of an initial denaturation step at 95 °C for 30 s, 35 cycles of 95 °C for 5 s, 55 °C for 30 s and 72 °C for 20 s. The melting curves for the PCR products were obtained by the stepwise increase of the temperature from 50 to 94 °C.