12 well plate

We estimated the biofilm formation of the Janthinobacterium sp. SLB01 strain grown in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) in LB broth at temperatures of 3, 22, and 30 °C. The biofilm amount was determined at regular time intervals according to a previously described method [11 (link)]. The wells of the plate were carefully emptied and washed twice with sterile 0.01 M phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) every 24 h. Then, 5 mL of an aqueous solution of 0.1% safranin (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at room temperature for 20 min. The dye was removed with 0.01 M PBS buffer. Then, the plates were inverted onto filter paper (Macherey-Nagel, Düren, Germany) and dried for 3 h. Safranin was extracted with a mixture of ethanol 96% and acetone (80:20 v/v). The biofilm formation was quantified by measuring the optical density (OD₄₈₄) of each sample using a spectrophotometer (GBC Scientific Equipment Ltd.—Cintra 20, Melbourne, Australia).

The Janthinobacterium sp. SLB01 strain was grown in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) with LB broth at temperatures of 3, 22, and 30 °C to assess the accumulation of violacein. Violacein was extracted according to the previously described technique [11 (link)]. Bacterial cells from a single well were harvested by centrifugation at 16,000× g for 15 min every 24 h. The cells were lysed with 10% sodium dodecyl sulfate (v/v) (AppliChem, Darmstadt, Germany), and incubated at room temperature for 5 min. Then, water-saturated butanol (1:2) was added and shaken. The upper phase, containing violacein, was separated from the aqueous phase by centrifugation at 16,000× g for 10 min. The extracted violacein was quantified using a spectrophotometer (OD₅₈₅) (GBC Scientific Equipment Ltd.—Cintra 20, Melbourne, Australia).

For MK and platelet experiments, 15–25 ml of whole blood was collected by venipuncture from two healthy controls, P3, P4 and P5 in the presence of 1.6 mg/ml EDTA. As access to patient blood was limited, these experiments were performed once (n = 1), with all samples processed in parallel. MKs were differentiated from whole blood samples using a protocol adapted from Balduini et al.^{66 (link)} Briefly, PBMCs were isolated from using Lymphosep (C C Pro, Oberdorla, Germany). Up to 2 × 10⁶ PBMCs were seeded per well in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) with StemSpan ACF medium (StemCell, Vancouver, Canada) supplemented with 10 ng/ml TPO, FLT3-L, IL-6, and IL-11 (all cytokines from Peprotech, Hamburg, Germany). On day 4, APEL2 medium (StemCell) supplemented with 5% Protein-free Hybridoma Medium II (PFHMII, Gibco by Life Technologies, Darmstadt, Germany) and the same cytokine composition was used. On days 1 or 2, 7, 10, and 14, cell morphology was assessed by microscopy and phenotype was analyzed by flow cytometry. For flow cytometry, cells were blocked with FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with CD42a-PE (BD Biosciences, Heidelberg, Germany), CD41-APC/Cy7, and CD61-APC (both Biolegend, San Diego, USA). Analysis was performed using a FACS Canto II and FACSDiva software v8.0.1 (BD Biosciences).

In order to investigate the regulation of the proteins involved in angiogenesis and inflammation processes, specific antibody arrays were performed using RayBiotech^®® C-Series Human Angiogenesis Array C1000 (code: AAH-ANG-1000, RayBiotech, Peachtree Corners, GA, USA) and RayBiotech^®® C-Series Human Inflammation Array C3 (code: AAH-INF-3, RayBiotech, Peachtree Corners, GA, USA). For this aim, PC3, DU145 and HUVEC cell lines (3.8 × 10⁵ cells) were seeded in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and kept overnight for attachment. For the detection of proteins, PC3 and DU145 cells were treated for 24 h at a concentration of Dt of 44.1 pM, while HUVEC cells were pre-treated for 1 h with tumor necrosis factor α (TNF-α) at a concentration of 10 ng mL⁻¹ before the treatment with 44.1 pM of Dt for 24 h.
After incubation, cell medium was collected. Protein concentration and purity were assessed using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For each condition tested, 1 mL of sample was used to perform specific antibody array, according to manufacturer’s protocol. Blots were analyzed using ImageLab software (Bio-Rad, Hercules, CA, USA) and results shown in terms of relative expression.

HEK293T cells (2 × 10⁵) were seeded in 12-well plates (Techno Plastic Products). The next
day, at a confluence of 50–70%, the cells were transiently
transfected with a mixture of DNA and PEI (8 μL PEI/1000 ng
DNA). Forty-eight hours post-transfection, the cells were washed with
1 mL PBS and lysed in 100 μL 1 × Passive lysis buffer (Promega).
The cells were lysed for 20 min on ice and centrifuged for 15 min
at 14 000 rpm to remove cell debris. The total protein concentration
in the supernatant was determined using the BCA assay.
Proteins
from the supernatant were separated on 12% SDS-PAGE gels (200 V, 45
min) and transferred to a nitrocellulose membrane (350 mA, 60 min).
Membrane blocking, antibody binding, and membrane washing were performed
using an iBind Flex Western device (ThermoFisher) according to the
manufacturer’s protocol. The primary antibodies were mouse-anti
Myc (Cell Signaling Technology 2276; diluted 1:2000). The secondary
antibodies were HRP-conjugated goat antimouse IgG, diluted 1:3000
(Jackson ImmunoResearch 115-035-003). The secondary antibodies were
detected with an ECL Western blotting detection reagent (Super Signal
West Femto; ThermoFisher) according to the manufacturer’s protocol.