Nonanal

To monitor the activity of the competence-specific promoter, PcomX, in response to CSP or XIP, luciferase reporter activity was assayed using a luxAB transcriptional reporter strain, S. mutans XT-Lx1 [34 (link)]. The bacterial growth and the luciferase reporter activity were assayed at regular intervals in a microtiter plate reader (Synergy HT, BioTek, USA), which read both bioluminescence and optical density at 600 nm. One percent of nonanal (Sigma-Aldrich) was used for the assay, since LuxAB-catalyzed luciferase activity requires the presence of nonanal as a substrate. Briefly, aliquots (300 μl) of cell cultures were transferred to the wells of a pre-warmed (37°C) microtiter plate. Aliquots (50 μl) of the solution containing 1% nonanal (diluted in mineral oil) in a volatile form were placed in the spaces between the wells of the microplate, which was then covered with the lip. The microtiter plate was placed in the pre-warmed reader for incubation and readings at every 15-min interval for continuous 5 hours. The results were expressed in relative luminescent units (RLU) divided by cell density of the cultures.

WST-1 assay was performed to assess cell proliferation. DPCs were seeded in a 96-well plate at a density of 3000 cells per well and treated with vehicle (DMSO; Sigma-Aldrich; St. Louis, MO, USA) or various concentrations of nonanal (Sigma-Aldrich). When required, vehicle (DMSO) or SQ22,536 (Sigma-Aldrich), a cAMP inhibitor, was added 1 h before nonanal treatment. Images of the cells were captured using a Nikon Eclipse TS2 microscope equipped with the DMX1200 camera (Nikon, Tokyo, Japan). Subsequently, the treated cells were incubated for 2 h with the WST-1 reagent (Sigma-Aldrich) diluted 1:10 in culture medium. Absorbance was read at 440 nm using the Infinite M200 microplate reader (Tecan; Männedorf, Switzerland). In all the experiments, the final DMSO concentration was less than 0.1% (v/v). Furthermore, to determine whether the proliferation of Hs68 was also affected by nonanal treatment, the same method was employed as above.

Trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA), propyl gallate, and sodium borate were purchased from Sigma Aldrich (Steinheim, Germany), and thiobarbituric acid (TBA) and sodium borohydride were purchased from Merck (Darmstadt, Germany). Chromatography-HPLC grade solvents, including chloroform, methanol, and acetonitrile, were purchased from Fisher Scientific (Schwerte, Germany), J.T. Baker (Gliwice, Poland), and Sigma Aldrich (Steinheim, Germany), respectively. The carboxymethyl-lysine standard was obtained from Cayman Chemical (Ann Arbor, MI, USA). The standard substances for volatile compound analysis, ethyl acetate, pentanal, hexanal, heptanal, octanal, nonanal, 2,4-decadienal, 2-heptanone, 1-pentanol, 1-hexanol, and 1-heptanol were purchased from Merck (Hohenbrunn, Germany), and the standard mix was purchased from Supelco (parrafine mix, 44585-U, Bellefonte, PA, USA).