Cl 1000 crosslinker

Cells were seeded at 1.0 × 10⁴ cells/well on 96-well plates, cultured overnight, and exposed to UV radiation or cisplatin (for 48 h) at varying doses. For UV radiation, cells were resuspended in PBS buffer, exposed to UV (254 nm) using a CL-1000 crosslinker (UVP, Upland, CA, USA), and incubated with fresh medium for 24 h. After treatment, cell viability was measured using CCK-8 (CK04; Dojindo, Kumamoto, Japan) following the manufacturer’s instructions.

Strains used in this study, and their sources are listed in Table 1. For preparations of live or UV inactivated S. aureus samples specifically, overnight cultures were diluted 1/100 in fresh warm TSB, incubated at 37°C while shaking at 200 rpm, and upon reaching OD₆₀₀ 0.5 test compounds in vehicle or pure vehicle were added to give a final concentration of 10 μg/mL. Cultures were grown to an OD₆₀₀ 1.7 and spun down. The supernatants were collected and frozen in 1 mL aliquots and the bacterial pellets were washed twice in sterile phosphate buffered saline solution (PBS). Washed bacteria were adjusted to OD₆₀₀ 0.5 in 20 mL PBS and 10 mL were frozen directly in 1 mL aliquots and the remaining 10 mL were subjected to UV radiation (λ = 254 nm; CL-1000 cross-linker; UVP, Cambridge, United Kingdom) by pulsed UV radiation of 6 sec per pulse for a total of 90 sec. Samples prior to UV and after UV were plated on TSA for number of colony-forming unit (CFU) analysis and multiplicity of infection (MOI) calculation, as well as for checking the viability after UV-irradiation. The Gram-positive bacteria Lactobacillus acidophilus NCFM (Danisco, Copenhagen, Denmark), and the Gram-negative bacteria Escherichia coli Nissle 1917 O6:K5:H1 (Statens Serum Institut, Copenhagen, Denmark) were grown as previously described [12 (link)].

Southern blotting was performed as described previously (Daniell et al., 2005 ; Southern, 2006 (link)). Briefly, tobacco DNA was digested using restriction enzymes Pfo I and Nde I, which cut regions within the flanking sequences used for homologous recombination. The hybridization probe was prepared using the PCR DIG Probe Synthesis Kit (Sigma-Aldrich) with primers (Supplementary Table 1) that generated a 1181 bp DIG-labeled probe from the homologous recombination regions. Digested DNA was separated on a 0.8% w/v agarose gel and transferred overnight to an Amersham Hybond membrane (GE Healthcare). The membrane was crosslinked at 1200V with a CL-1000 Crosslinker (UVP) and hybridized with the probe overnight at 50°C in DIG Easy Hyb buffer (Roche). After two washes in 2 × Saline Sodium Citrate (SSC, 20 × SSC is 3.0 M NaCl, 0.3 M sodium citrate, pH = 7.0) buffer + 0.1% SDS at room temperature and three washes in 0.5 × SSC + 0.1% SDS at 68°C, membrane was blocked with DIG blocking buffer (Roche). Hybridized probe was detected by soaking membrane in Anti-Digoxigenin-AP Fab antibody solution (Roche) followed by CSPD chemiluminescent substrate (Roche) detection. A MicroChemi 4.2 Bio Imaging System was used to visualize the bands after one hr exposure.