Percentage viability was calculated as the number of viable cells after treatment divided by the total number of cells without peptide, times 100. Overnight cultures in THYE were pelleted, washed, resuspended in sterile 1× PBS, and diluted 1 : 100 using warm
CDM. Each suspension was supplemented with either 1% DMSO or 10 μM XIP and used to inoculate polystyrene plates. After 24-h incubation, the biofilms were dried and strained with 0.1% Safranin Red. Overnight cultures of UA159 check details and its derivatives were diluted 20× in fresh THYE or CDM and grown to an OD600 of 0.4–0.5 in the presence or absence of 0.4 μM CSP or 10 μM XIP, respectively. For growth in CDM, overnight cells were washed and resuspended in 1× PBS prior to inoculation and harvesting. Controls included THYE without added peptide, as well as CDM with 1% DMSO. RNA isolation, DNAse treatment, cDNA synthesis, qRT-PCR, and expression analyses were carried out as previously described (Senadheera et al., 2005). Primers used for qRT-PCR are as follows: comR (For: CGTTTAGGAGTGACGCTTGG, Rev: TGTTGGTCGCCATAGGTTG), comS (For: TTTTGATGGGTCTTGACTGG, Rev: TTTATTACTGTGCCGTGTTAGC) and comX (For: ACTGTTTGTCAAGTCGCGG Rev: TGCTCTCCTGCTACCAAGCG). Expression was normalized to that of 16SrRNA gene, and statistical analyses were performed on four independent experiments using Student’s Trametinib datasheet t-test (P < 0.05). Overnight cultures in CDM were
diluted 100-fold and grown for 48 h at 37 °C in 5% CO2 air mixture. Cell-free supernatants were obtained by centrifugation and filter sterilized using a 0.45-μm syringe filter. Samples were lyophilized and,
once dry, reconstituted in 2 mL of 5% MeOH/H2O (v/v) prior to analysis by HPLC-ESI-MS/MS (Dionex UltiMate 3000 HPLC system with variable UV detection in line to a Bruker amaZon X ion-trap mass spectrometer operating in positive ionization mode with auto MS/MS enabled). Analytical scale analysis was performed on a 250 × 4.60 mm Phenomenex Luna 5μ C18(2) 100 Å column (Serial no. 516161-20) with a flow rate of 1 mL min−1 and the following program consisting of solvents A (water + 0.1% formic Cell press acid) and B (acetonitrile + 0.1% formic acid): 0–2 min, equilibration at 5% B; 2–18 min, linear gradient to 100% B; 18–20 min, constant 100% B, 20–20.5 min, linear decrease to 5% B; 20.5–23 min re-equilibration at 5% B. The identity of XIP in culture supernatants was confirmed by comparison with the retention time and MS/MS fragmentation of sXIP. To quantify XIP levels, a directed LC-MS/MS experiment was performed using selected-reaction monitoring (SRM) MS/MS. The SRM m/z transition 876.4 658.4 was monitored, corresponding to a –SL loss from the GLDWWSL parent ion, generating a GLDWW daughter ion. Resulting peak areas were integrated, and final concentrations calculated from a linear calibration curve created using CDM spiked with sXIP and processed in an identical way to cell free supernatants.