Visionworks software

Cells with or without MILIP knockdown were grown to 90% confluence, and then monolayers were scratched with 200 µl pipette tips across the center of wells. After being washed with PBS for three times, cells were then maintained in the serum-free medium and imaged over a 24 h period at 12 h intervals to monitor wound width. The wound width was quantified using the Vision Works software (Analytik Jena, Jena, Germany).

Western blot analysis of kidney tissue homogenates was performed to analyze SNAIL1, TWIST1, SMAD3, and SMAD7 protein expression (Invitrogen, Waltham, MA, USA). According to our previous real-time PCR analysis, GAPDH (Invitrogen, Waltham, MA, USA) was also used as a housekeeping protein. Proteins were extracted from kidney tissue using P-TER solution (Thermo Fisher Scientific, USA) and quantified by the BCA protein assay kit (Thermo Fisher Scientific, USA). A quantity of 50 µg of total proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). Blocking was performed using 5% non-fat dry milk in 1X TBST for 1h at 4 °C with constant agitation. Membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000 (SNAIL1, TWIST1, SMAD3, and GAPDH) and 1:500 (SMAD7) in 1X TBST. Antibody binding was revealed with an HRP-conjugated secondary anti-antibody diluted 1:5000 in 1X TBST using a BM Chemiluminescence kit (Roche Diagnostics, Indianapolis Ind, Indianapolis, IN, USA). Densitometric analysis was performed with a UVP ChemiStudio image analyzer (Analytik Jena, Jena, Germany) using the VisionWorks^® software (Analytik Jena, Germany). The semiquantitative analysis of every protein was shown as normalized levels.

For dual-species competition assays between each S. mutans strain (UA159, ΔlrgAB, SAB161, or SAB163) and S. gordonii strain (DL1 or ΔspxB), 200 μl of each competing seed culture (one from S. mutans and the other from S. gordonii), were each adjusted to OD₆₀₀ = 0.5 in BHI medium, centrifuged, the pellet washed with PBS (phosphate buffer saline), resuspended in 200 μl PBS, and then diluted 1:100 into 200 μl fresh medium in individual wells of a 96-well plate (Costar^TM3595, Corning). After 24 h incubation at 37°C in an aerobic atmosphere, 5 μl culture from each well was inoculated on Prussian blue agar plates (Saito et al., 2007 (link)), indicating the production of H₂O₂. The plates were incubated for an additional 24 h at 37°C in an aerobic atmosphere. Growth and blue precipitation zones were documented and analyzed with VisionWorks^® software (Analytik Jena, Upland, CA, United States). Original grayscale .tif images of each plate were each converted to RGB color, and mid-tone color balanced (−50, 0, +31) in Adobe Photoshop 21.2.1 to improve the contrast and clarity of each image.