Gen5 program

The ALDH1 ELISA kit (ab155894, Abcam, Cambridge, MA, USA) was used to measure ALDH1 activity in BON and QGP-1 cells with different treatments. The reaction mixture of ALDH assay buffer and ALDH substrate was incubated for 60 min at 22 °C. Fluorescence values were recorded at Ex/Em 535/587 nm by the Gen5 program (BioTek, Winooski, VT, USA) and used to calculate ALDH activity, based upon the manufacturer’s instruction.

B. bruxellensis LAMAP2480 strain was grown in 5ml of SD minimum medium (2% w/v glucose and 6.7g/l yeast nitrogen base (YNB; Difco Laboratories, Detroit, United States)) at 28°C until saturation (stationary phase). Then, 1×10⁵ cells/ml were inoculated in 200μl of the same medium in the absence and presence of 100mg/l of pCA (Sigma-Aldrich, Inc.; United States) in triplicate on a Cell Culture Plate (SPL Life Sciences, Korea). The microplates were incubated at 28°C in the dark and at different light intensities using white fluorescent lamps at a light intensity of 2,500 lux and 4,000 lux. Varying light intensities were provided by adjusting the light with the help of Lux meter UNI-T UT 382 USB (Dongguan, China). Absorbance measurements were made at 600nm for 10days in the Epoch^™ equipment (BioTek, United States), coupled to the Gen5 program (BioTek, United States).
The specific growth rate was determined by the slope of the exponential growth phase according to the equation xt=x0+μt, where xt and x0 represent the biomass in optical density (OD) at time t (h) and t=0, respectively (Barata et al., 2008 (link)). The lag phase was determined as described by Buchanan and Cygnarowicz (1990) (link). All experiments were performed in triplicate.

The chemiluminescence analysis of lucigenin derivatives was used to determine the activity of NADPH oxidase in a suspension of membrane preparations of HL-60 cells. For this, 100 mg of total protein from the suspensions of the membrane preparations were placed in a 96-well microplate at a final volume of 200 µL with PBS and incubated at 37 °C for 30 min with the acrylonitrile derivatives. VAS2870 (20 µM) was used as a control of inhibition. Chemiluminescence was measured for 1 h at 2 min intervals with a Synnergy H1 hybrid luminometer multi-mode microplate reader and GEN5 program (Biotek Instruments, Inc., Winooski, VT, USA). The reaction started with the addition of 100 μM NADPH to the cell suspension (200 μL: 100 μg protein + PBS) containing 100 μM lucigenin. The activity was expressed as relative units of luminescence per second, relative to the control.

Surface area measurements (µm²) and micrographs of at least five spheroids per treatment point were recorded from day three (immediately before the BP treatment = time 0) and after 24 and 96 h of post-treatment. Spheroids were exposed to single compounds at concentrations 10, 20, 40, and 80 µM and 1, 2, 4, and 8 µM for 24 and 96 h, respectively, and their combinations at concentrations of 10 + 10 µM, 20 + 20 µM, and 40 + 40 µM and 1 + 1 µM, 2 + 2 µM, and 4 + 4 µM for 24 and 96 h, respectively (see Table 2). Average surface area measurements and micrographs were captured using Cytation 5 (BioTek, Winooski, VT, USA) with a built-in microscope and a wide-angle-view camera at 4× magnification, and analysis was performed using the Gen5 program (Software for imaging and microscopy, BioTek, USA, version 3.11). Experiments were repeated three times in independent biological replicates.
In each experiment, a negative control (cell medium), solvent control (cells exposed to 0.3% and 0.06% DMSO for 24 and 96 h, respectively), and positive control (15% DMSO) were included. Graphical and statistical analyses were performed using GraphPad 8 software by one-way analysis of variance (ANOVA) test with a Dunnett post hoc [p < 0.05 (*), p < 0.001 (**), p < 0.0001 (***) were considered statistically significant].

A recombinant strain of HSV-1 (KOS-GFP-HSV-1) was used for all experiments. This strain facilitated the detection of infected cells via GFP reporter expression (Figure 5A). At 24 h post-infection, the Cytation 5 was used to image and quantify GFP-expressing cells (Figure 5B). Image analysis was performed using the Gen5 program (version 3.11) (Agilent Technologies, Santa Clara, CA, USA). Images were acquired using a 4x magnification lens with the brightfield high contrast and GFP channels. Total cells, GFP+ cells, and the mean fluorescent intensity (MFI) of each well were quantified from images captured at 4x magnification using the Cytation 5. The percentage of GFP+ cells was calculated by dividing the number of GFP+ cells by the total cell count for each image.

Wound healing assay studies changes in cell migration and proliferation under pro-inflammatory conditions. Confluent HaCaT cells grown on the 96-well plate were used for the assay. Half of the plates was treated with mitomycin at a final concentration of 1 μg/ml for 2 h to stop the proliferation and observe only migration. After PBS wash, the wounds were made by BioTek AutoScratch Wound Making Tool (Agilent, USA), the cells were washed again, supplemented with HaCaT culture medium, and treated with LPS as mentioned above. The cells were maintained in BioTek BioSpa 8 Automated Incubator (Agilent) and photos of the wounds were acquired by BioTek Cytation 5 Imaging Reader (Agilent) each 6 h for 24 h. The area of the wounds was calculated in the Gen5 program (Agilent).