Lumistar omega

Luciferase-labeled DIPG cells were plated on top of purified microglia or astrocytes (670 cells/well), in 0.3-cm² wells. Following a 72-h incubation, luciferase activity was measured using LUMIstar® Omega (BMG LABTECH, Ortenberg, Germany) microplate luminescence reader. Each experiment consisted of a minimum of 4 wells per condition, and each experiment was repeated at least 6 times per cell line.

To analyze the induction of apoptosis, the activity of effector caspase-3 and -7 was measured using a Caspase-Glo® 3/7 assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Cells were transiently transfected in four technical replicates in 96-well plates with 3 × 10³ SUM159 and 5 × 10³ MDA-MB-231 cells per well using HiPerFect Transfection Reagent (Qiagen) according to the reverse transfection protocol. After 48 h, the luminogenic reagent was added to the cells as instructed in the protocol, and signals were measured with a LUMIstar Omega (BMG LabTech).

Efflux of rhodamine 6G (R6G) was carried out as described [18 (link)]. Briefly, cells were grown overnight in YEPD and were diluted in 5 mL YEPD to grow at 30 °C under constant agitation until a density of 2 × 10⁷ cells/mL was obtained. Cells were centrifuged, washed, and resuspended in 2 mL PBS (pH 7). Energy deprivation was next achieved by 1 h incubation in PBS at 30 °C. R6G was then added at a concentration of 10 µg/mL and the incubation was continued for 1 h. After this incubation time, cells were centrifuged, washed with PBS at 4 °C, and resuspended in a final volume of 200 µL PBS. Fifty microliters of individual strains were diluted in 50 µL PBS and aliquoted in a 96-well microtiter plate, which was placed in a LUMIstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany) with temperature control at 30 °C. Baseline fluorescence emission (excitation wavelength: 340 nm; emission wavelength: 555 nm) was recorded as relative fluorescence units (RFU) for 5 min and glucose (1% final concentration) was next injected to initiate R6G efflux. As a control, no glucose was added to separate aliquots of each strain. Data points were recorded in duplicates for 60 min at 1 min intervals and were plotted in Graph Prism software (Version 9.1.0, GraphPad Software, San Diego, CA, USA).

Keratinocyte activation by the Keap1/Nrf2-ARE (antioxidant response element) pathway was measured by the KeratinoSens^TM assay [48 ]. The assay uses an antioxidant response element (ARE)-coupled luciferase to sense Nrf2 activation. The KeratinoSens™ cell line was obtained from Givaudan (Vernier, Switzerland). The cells were cultured in DMEM supplemented with Glutamax^TM, Fetal Calf Serum (FCS) 9.1%, and Geneticin^TM (500 μg/mL) at 37 °C in an atmosphere of 5% CO₂ and 95% humidity. Cells were seeded on 96-well plates until reaching 80% confluency. Cells were then incubated with obacunone for 48 h in antibiotic-free DMEM. At the end of the incubation period, cells were washed and lysed for 20 min. Then, Promega firefly luciferase reagent was added, and the luminescence was immediately measured on a Lumistar plate reader (Lumistar Omega, BMG Labtech). An increase in luciferase activity in sample-treated cells was calculated in comparison to DMSO-treated cells (negative control) and expressed as fold luciferase activity induction (Imax). Following the manufacturer’s instructions, activation of the Nrf2 pathway was considered positive when the Imax was equal to or higher than 1.5 and statistically significant compared to the negative control.

The anti-biofilm activity assay of VP_AHPND was determined as described previously with slight modifications (Nesper et al., 2001 (link)). Briefly, an overnight VP_AHPND culture in LB broth was added to a 96-well microtiter plate with or without V. alginolyticus extract (2.5, 5, and 10%) and incubated at 30 °C for 24 h. Planktonic cells were removed by washing the wells with sterile water, fixed with 2.5% glutaraldehyde, and stained with 0.4% (w/v) crystal violet. After that ethanol-acetone (80:20, v/v) was added. The OD was determined at 570 nm in a microplate reader (LUMIstar Omega, BMG LABTECH, Germany).
The effect of V. alginolyticus extract on EPSs in biofilm was visualized by scanning electron microscopy (SEM) as described previously with minor modifications (Singh, Mishra & Jha, 2017 (link)). Briefly, biofilms of VP_AHPND grown on glass coverslips (1.0 × 1.0 cm) submerged in LB broth with or without V. alginolyticus extract in 24-well plate were gently washed with phosphate buffer saline (PBS) to remove planktonic cells. Afterwards the cells were fixed with 2.5% glutaraldehyde (v/v) at 4 °C for 12 h. After dehydrating in a gradient ethanol series (30–100%, v/v) for 15 min each, the dried samples were coated with gold and investigated under a SEM (Quanta 400, Thermo Fisher Scientific, USA).

PP-spheroids (HUES4) at passage 3 were dissociated as described above. A Bravo liquid handling robot (Agilent) was used to deposit 8 µl of cell suspension in all but 3 wells of a 96-well OptiPlate (PerkinElmer). GFR-Matrigel mixture without cells was added to the last three wells, to serve as a negative control. For the first 48 h, PP-spheroids were cultured as normal, in an expansion medium. After 48 h expansion medium was removed, wells were washed with 1× PBS twice, and different media were added in triplicates, including 26 conditions and 3 controls (details on Supplementary Table 3). The medium was again changed at day 5 and 8. After a total of 10 days in culture, Cell Titer-Glo luciferase assay (Promega) was performed according to the manufacturer’s instructions, and luminosity was measured in the LUMIstar omega (BMG Labtech). The validation experiments were performed in Nunc 4 well plates with both the HUES4 and SBAD3.4 cell lines, with the same timeline used in the original screening experiment, except for Infigratinib, which was only added from day 9–10. Cells were collected at days 5 and 10 of culture for EdU quantification. Validation in fetal spheres was performed in 8-well µ-slides (Ibidi).

Forty-eight hours post-transfection of the HEK293FT cells with the corresponding plasmids (see transient transfection assays), the DMEM complete media was replaced with 5 μM coelenterazine h (Promega) in calcium- and magnesium-free Hank’s Balanced Salt Solution (HBSS). The BRET measurements were immediately taken at 37 °C using a LUMIstar Omega plate reader (BMG Labtech Mornington, Vic, Australia). Filtered light emissions were simultaneously measured at 460–490 nm for RLuc8 and 520–550 nm for Venus. The BRET ratio was calculated by subtracting the ratio of 520–550 nm emission and the 460–490 nm emission^{57 (link),58 (link)}.