1 mm glass beads

For western blotting, 10 larvae (3dpf) of each genotype were collected. Larvae were then lysed in 100μL of OCG buffer (0.3M NaCl, 2.5μM EDTA pH8, 0.9M Tris HCl pH7.5, protease inhibitors, phosphatase inhibitor) with 1mm glass beads (BioSpec) for 3x20sec in the sonicator Bioruptor (diagenode). Qubit protein assay kit (Invitrogen) was used to obtain protein concentration. Proteins (25μg) were resolved on a Bolt 10% Bis-Tris Plus Gel (Invitrogen), blotted onto nitrocellulose membrane using iBlot2 transfer stacks (Life Technologies) according to manufacturer’s protocol. Proteins were probed with rabbit anti-human Cx43 (1:2000) (Sigma-Aldrich) and rabbit anti-β-Tubulin antibodies (1:2000) (Abcam) after saturation in PBT (PBS, 0.1% Tween-20) containing 5% of milk. Proteins were then revealed using an enhanced chemiluminescence detection system (Pierce ECL Plus Western Blotting Substrate, Invitrogen) with goat anti-rabbit HRP antibody (1:2000) (Abcam).

Plasma cholesterol and triglycerides were measured via an enzymatic colorimetric assay according to the manufacturer’s protocol (Cholesterol Liquicolor CHOD_PAD; Human #10028, Instruchemie, Delfzijl) (Sigma Triglyceride (GPO Trinder) kit (Sigma Tr0100)). Absorbance was measured with the BioRad Benchmark Plate Reader (170-6750XTU; Bio-Rad, Hercules, CA). To measure liver cholesterol and triglycerides, liver homogenates were made. Approximately 40–50 mg of frozen liver tissue was homogenized in 1 ml SET buffer (250 mM Sucrose, 2 mM EDTA, 10 mMTris) with 1 mm glass beads (Biospec, art. 11079110) on the maximal setting of the Biospec Mini Bead Beater-1. Afterwards, samples underwent two freeze-thaw cycles for complete cell destruction. To optimize cell destruction, samples were taken through a 25Gx5/8” needle several times and a final thaw cycle was added. Total protein content was measured via bicinchoninic acid (BCA) assay (23225; Pierce, Rockford, IL). Liver cholesterol and triglycerides were measured via the enzymatic colorimetric assay.

Passiflora hybrid seeds were soaked in water for 24 h and embryos were excised by removing the seed coat and endosperm under a dissecting microscope. The excised embryos were rinsed in sterile water and stored dry at −80 °C until further use. Stored embryos were suspended in 88 μL of 50 mM NaOH and homogenized for 1 min in the presence of five to six 1 mm glass beads (BioSpec Inc., Bartlesville, OK, USA) using Mini-BeadBeater-96 (Glen Mills Inc., Clifton, NJ, USA). The disrupted embryos were incubated at 95 °C for 10 min and neutralized by adding 12 μL of 1 M Tris-HCl (pH 8.0). The crude extract was vortexed, briefly centrifuged and 5 μL of supernatant was used for PCR.
Plastid inheritance in hybrid embryos was assessed by sampling 10 seeds per cross for 44 interspecific crosses. The PCR amplification of crude embryo extract was carried out using Seed-Direct^TM PCR mix (D300, Lamda Biotech, St. Louis, MO, USA). The PCR reaction contained 5 μL of crude extract, 10 μL of 2× Seed-Direct^TM PCR mix, 1 μL of 10 μM forward and reverse primers and 7 μL of H₂O. The PCR program included 5 min at 95 °C, followed by 34 cycles of 30 s at 95 °C, 30 s at 52–56 °C, and 1 min at 72 °C with final extension of 5 min at 72 °C. Annealing temperatures were adjusted according to the melting temperature of the different primer sets.

For quantitation of viable B. cereus, harvested, infected, and/or treated eyes from euthanized mice were homogenized in 400 µL PBS with sterile 1-mm glass beads (BioSpec Products, Inc., Bartlesville, OK, USA). An amount of 20 µL of each eye homogenate was track diluted 10-fold in PBS. Dilutions and the remaining homogenates were plated onto BHI agar plates for quantitation (50 (link)). The limit of detection for quantifying B. cereus in eye homogenates was 1 CFU.
For histology, infected and/or treated eyes were harvested from euthanized mice, incubated in high-alcoholic fixative for 4 hours, and then transferred to 70% ethanol. Eyes were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (33 (link)).

Upon harvest, females were freeze-killed at -80°C and transferred on ice. Midguts were dissected in 1X phosphate-buffered saline (PBS) under 10X magnification. Forceps were decontaminated between each individual using Surfa’Safe (Anios) to prevent cross contamination. Individual midguts were immediately homogenized for 30 sec at 6,000 rpm in tubes (VWR) containing ~20 1-mm glass beads (BioSpec) in 800 μL of TRIzol (Life Technologies) and stored at -80°C. Samples were thawed at room temperature (20–25°C) and 150 μL of chloroform (Sigma-Aldrich) were added followed by vortexing for 30 sec. After a 5-min incubation at 4°C, samples were centrifuged at 4°C for 15 min at 14,000 rpm. The upper aqueous phase was harvested and transferred to a cold tube containing 400 μL of 2-propanol (Sigma-Aldrich) supplemented with 1 μL GlycoBLUE (Ambion, Life Technologies). Samples were incubated at -20°C overnight and centrifuged at 4°C for 15 min at 14,000 rpm to pellet RNA. The pellet was washed with 800 μL of 70% ice-cold ethanol (Sigma-Aldrich) at 4°C for 10 min at 14,000 rpm, and allowed to dry for 10 min at 37°C. Total RNA was resuspended in 6 μL, of which 1 μl was diluted into 9 μl of RNase-free water for DENV quantification by RT-qPCR, while the remaining 5 μl were used for transcriptome sequencing. All the samples were stored at -80°C until use.

V. cholerae strains were grown overnight at 37 °C in LB liquid medium with shaking. Cell clusters were dispersed by vortex for 1 min with 1 mm glass beads (Biospec). The resulting cell suspensions were back diluted 100-fold and incubated for an additional 2 h with shaking at 37 °C in LB medium so that the cultures reached early exponential phase (OD₆₀₀ = 0.1-0.2). Following another 1 min of vortex with beads, cultures were diluted with LB medium to yield inoculum cell densities ranging from OD₆₀₀ = 0.001 to 0.1. When higher inoculum seeding densities were necessary, the growth time following resuspension was increased accordingly. A total of 5.5 mL of culture inoculum was added to wells of 12-well plates so that the depth of liquid in each well was 14 mm. The cultures in the wells were overlaid with sterile mineral oil (Sigma-Aldrich, volume 850 μL) to prevent evaporation and pellicle desiccation. Pellicles were allowed to form at room temperature (20 to 22 °C) and imaged from 15 h to 48 h as specified.