Falcon primaria

To analyze tumor cell adhesion, HUVECs were transferred to six-well multiplates (Falcon Primaria; BD Biosciences, Franklin Lakes, NJ, USA) in complete HUVEC medium. When confluency was reached, PC3^par or PC3^res cells were detached from the culture flasks by accutase treatment (PAA Laboratories, Cölbe, Germany) and 0.5 × 10⁶ cells were then added to the HUVEC monolayer for 30, 60, or 120 min. Subsequently, non-adherent tumor cells were washed off using warmed (37 °C) Medium 199. The remaining cells were fixed with 1% glutaraldehyde. Adherent tumor cells (appearing as rounded, light cells) were counted in five different fields (5 × 0.25 mm²) using a phase contrast microscope and the mean cellular adhesion rate was calculated.

To analyse tumour cell adhesion, HUVEC were transferred to 6-well multiplates (Falcon Primaria; BD Biosciences) in complete HUVEC-medium. When confluency was reached, treated versus non-treated PC-3, DU-145 and LNCaP cells were detached from the culture flasks by accutase (PAA Laboratories, Cölbe, Germany) and 0.5 × 10⁶ cells were then added to the HUVEC monolayer for 1, 2 or 4 hrs. Subsequently, non-adherent tumour cells were washed off using warmed (37°C) Medium 199. The remaining cells were fixed with 1% glutaraldehyde. Adherent tumour cells were counted in five different fields of a defined size (5 × 0.25 mm²), using a phase contrast microscope, and the mean cellular adhesion rate was calculated.

To analyze Caki-1 adhesion, HUVECs were seeded onto six-well multiplates (Falcon Primaria; BD Biosciences, Heidelberg, Germany) in complete HUVEC medium. When confluency was reached, Caki-1 cells (everolimus-resistant and -sensitive, SFN treated and non-treated controls) were detached from their culture flasks by Accutase treatment (PAA Laboratories, Cölbe, Germany). Tumor cells (0.5 × 10⁶) were then added to the HUVEC monolayer for 30, 60, or 120 minutes. Subsequently, non-adherent cells were washed off using warmed (37°C) M199. Adherent cells were fixed with 1% glutaraldehyde and counted in five different fields, each 0.25 mm² , using a phase-contrast microscope. Mean cellular adhesion in the five fields was calculated.

Primary swine macrophage cell cultures were derived from pig peripheral blood and were prepared as previously described [21 (link)] using an existing collection of swine blood approved IACUC protocol 205.02-17-R: Reagent Production for the Molecular Pathogenesis of Classical Swine Fever Virus (CSFV) and African Swine Fever Virus (ASFV). Macrophages were seeded in 6-well plates (Primaria Falcon, Becton Dickinson, Franklin Lakes, NY). ASFV Georgia 2007 strain [22 (link)] was used in the macrophage infection at MOI = 1. ASFV infection experiments were conducted with three biological replicates using three different animals as the source of macrophages. Mock infection was also performed in the cultured macrophages from the three commercial domestic pigs as non-infected controls.

Primary swine macrophage cell cultures were derived from pig peripheral blood as previously described [110 (link)]. Macrophages were seeded in 6-well plates (Primaria Falcon, Becton Dickinson, Franklin Lakes, NY, USA). The VSV strains used in this study included (a) NJ0612NME6, an epidemic VS New Jersey virus (VSNJV) strain that caused outbreaks in the US from 2012 to 2014 and was isolated from a naturally infected equine in New Mexico in 2012 and (b) NJ0806VCB, a VSNJV strain that circulated in 2006 in an endemic area of Mexico and was obtained from a naturally infected bovine in Veracruz [111 (link)]. These viruses have an overall nucleotide identity of 99.08%, and differences in their pathogenesis were reported in a previous study, which indicated that NJ0612NME6 has higher virulence than NJ0806VCB in pigs [12 (link)]. VSV infection experiments were conducted with three biological replicates using ex vivo cultured primary macrophages isolated from three different commercial domestic pigs. Macrophages were infected with an MOI of 10 TCID₅₀ of each virus. Mock infection was also performed in the cultured macrophages from these same pigs as non-infected controls.