0.45 μm pore size filter

An overview of cell free supernatants utilised in this study is provided in Supplementary Fig. 1. EcN Supernatants (EcN-SNT): Supernatants derived solely from E. coli Nissle 1917 cultures (EcN-SNT) were obtained by recovery of EcN cultures from LB broth cultures by centrifugation, washing of recovered cells with PBS, and re-suspension at 2 × 10⁹ Cfu/mL in Krebs buffer. These cell suspensions were incubated at 37 °C, 5% CO₂ for 1 h. Supernatants were then collected by centrifugation (13,000 g, 5 min) and filtration using a Whatman 0.45 μm pore-size filter, before use directly in experiments. Ileum Supernatants (I-SNT): Supernatants obtained from untreated ileum sections were recovered following incubation of tissue in Krebs buffer (37 °C, 5% CO₂ for 1 h), followed by centrifugation and filtration as above. Ileum EcN modified Supernatants (I-EcN-SNT):To obtain supernatants exposed to both Ileum and EcN cells independently, EcN cells recovered from LB broth cultures were suspended in I-SNT preparations described above to a final density of 2 × 10⁹ Cfu/mL, and incubated for a further hour (37 °C, 5% CO₂). The resulting ileum-EcN-SNT was recovered by centrifugation and filtration as above, before use in further experiments.

KSHV inoculum was obtained from BCBL-1 cells (5 x 10⁵ cells/ml) induced with 20 ng of TPA (12-O-tetradecanoylphorbol-13-acetate; Sigma)/ml as described previously [27 (link)]. After 5 days, cells were pelleted, and the supernatant was run through a 0.45-μm-pore-size filter (Whatman). Virions were pelleted at 30,000xg for 2 h in a JA-14 rotor, Avanti-J-25 centrifuge (Beckman Coulter). The viral pellet was resuspended in EBM-2 without supplements.
The recombinant KSHV BAC16 originally made in the Jung lab [41 (link)] was obtained from the Renne lab (see acknowledgements). The KSHV BAC16 mutant viruses were obtained from the Renne lab and are described elsewhere [35 (link)–36 (link)]. BAC16 and mutant viruses were passaged in iSLK cells as described [35 (link)]. In addition, viruses were confirmed for their mutation by PCR and sequencing. Lytic replication was induced by adding 1 μg/mL of doxycycline and 1 mM of sodium butyrate. Virus was harvested from the supernatant 4 days post induction as described above.
KSHV infections of primary hDMVEC were performed in serum-free EBM-2 supplemented with 8ug/ml polybrene for 3 hours, after which the medium was replaced with complete EGM-2. Mock infections were performed identically except that concentrated virus was omitted from the inoculum.

Extracellular KSHV particles were obtained from BCBL-1 cells (5 x 10⁵ cells/mL) induced with 20ng of TPA (12-O-tetradecanoylphorbol-13-acetate; Sigma)/mL as described previously. After 5 days, cells were pelleted, and the supernatant was run through a 0.45μm-pore-size filter (Whatman). Virions were pelleted at 30,000xg for 2 h in a JA-14 rotor, Avanti-J-25 centrifuge (Beckman Coulter). The viral pellet was resuspended in EBM-2 without supplements. The KSHV Bac16 viruses were made as described previously [42 (link),53 (link)]. The ad-GFP and KLAR viruses were made as described previously [42 (link)].
KSHV infections of all cell types were performed in serum-free EBM-2 supplemented with 8μg/mL polybrene for 4 h, after which the medium was replaced with complete EGM-2. Mock infections were performed identically except that concentrated virus was omitted from the inoculum. Infections with ad-GFP and KLAR were performed the same as above except serum-free EBM-2 was supplemented with 1μg/mL poly-L-lysine instead of polybrene.