Six well polystyrene plate

Trans‐well co‐culturing was performed using six‐well polystyrene plates (Corning, Inc) and inserts with permeable membranes having a pore size of 0.4 µm. HRMC were seeded into the lower chamber, and HK‐2 cells in the upper chamber (insert). Stable HK‐2 cell lines with control lentivirus or depletion, were cultured in hypoxic conditions for 4 hours, and then were moved into the wells containing HRMC and both cell types were co‐cultured in reoxygenation for 24 hours. HRMC were digested for testing. The experimental groups were as follows: (a) HRMC co‐cultured with HK‐2 cells infected with control lentivirus, (b) HRMC co‐cultured with HK‐2 cells infected with IRE1α or JNK1 knock‐down lentivirus, (c) HRMC co‐cultured with H/R HK‐2 cells infected with control lentivirus, (d) HRMC co‐cultured with H/R HK‐2 cells infected with IRE1α or JNK1 knock‐down lentivirus.

A semi-static biofilm model was used to assess biofilm formation of S1 and S2, as described before[43 (link)]. Overnight bacterial cultures were diluted to an OD660 of 0.01 in 6 ml LB medium with 1%(wt/vol) glucose and added to a coverslip coated with poly-L-lysine (0.45 μm; diameter, 12 mm; Becton Dickinson) inside a well from a six-well polystyrene plate (Corning Inc.). Biofilms were grown at 30°C for 48 hat 120 rpm. After 48 h, the coverslips were washed with 0.85% (wt/vol) NaCl and the biofilms were chemically fixed with 8% (vol/vol) glutaraldehyde (Merck) for 20 min. Subsequently, the biofilms were stained with 15 μg/ml propidium iodide (PI) in 0.85% (wt/vol) NaCl that was removed after 15 min. The coverslips were transferred to glass microscope slides and analyzed by a confocal laser scanning microscope (CLSM) (Leica SP5), equipped with an oil plan-Neofluar ×63/1.4 objective. PI was excited at 488 nm. Z stacks were taken with an interval of 0.42 μm. Pictures were analyzed with LAS AF software (Leica), and biofilm thickness and biomass were quantified using Comstat[44 (link)]/Matlab R2013b software (the MathWorks). The average thickness and biomass of the biofilms were measured at ten randomly chosen positions and statistical significance determined by unpaired two-tailed Student's t-test. Experiments were performed twice in duplicate.

S. mutans UA159 was inoculated with S. gordonii DL1 using a two-compartment system [17 (link), 18 ] with a slight modification. Briefly, each well of a six-well polystyrene plate (Corning Inc., Corning, NY) was separated into two compartments using Nunc 25-mm Tissue Culture Inserts with 0.2-μl Anopore membranes (Nunc, Roskilde, Denmark). Each compartment contained CDM supplemented with 0.5% sucrose. S. gordonii DL1 was inoculated in the upper compartment, and S. mutans UA159 was inoculated in the lower layer. As controls, S. mutans was inoculated in both the upper and lower compartments. Each overnight culture was added to 3 ml CDM (culture:CDM = 1:30) and incubated at 37°C under anaerobic conditions for 24 h. The S. mutans biofilm in the lower compartment was then collected for analysis.

Samples to be subjected to two-dimensional gel electrophoresis (2-DE) were prepared using both chemical and mechanical extraction to ensure high yield and optimum solubility of whole-cell proteins. Biofilm cells on a six-well polystyrene plate (Corning) were harvested with a cell scraper (Asahi Glass, Tokyo, Japan) and washed four times with distilled water. The bacterial biofilm was suspended with 1.0 ml distilled water and disrupted using a Mini-Bead Beater (Biospec Products, Bartlesville, OK) with a 2 ml tube containing 0.1-mm-diameter silica sphere beads (Lysing Matrix B; MP Biomedicals LLC, Solon, OH) at 4800 rpm for 30 s. After disruption, the samples were cooled on ice for 3 min. This procedure was repeated five times. The aliquots were transferred to 1.5 mL tubes and centrifuged at 15,000 rpm for 5 min. The protein concentrations of the supernatant were measured (Quick Start Bradford Dye Reagent 1×; Bio-Rad, Hercules, CA) and the supernatant was subjected to acetone precipitation. A total of 200 μg protein per 1.5 ml tube was precipitated with 1.0 ml acetone and incubated at −30°C for more than 10 min. The samples were centrifuged at 15,000 rpm for 5 min, and the acetone was removed. Sample preparation was also performed with a 2-D Clean-Up Kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), according to the manufacturer’s instructions.