For maintenance, iSLK cells were cultured in growth medium of DMEM/fetal calf serum 10% containing 1 μg/mL puromycin (Fujifilm-Wako Chemicals, Osaka, Japan) and 0.25 mg/mL of G418 (Fujifilm-Wako Chemicals). KSHV BAC16 mutant (∆ORF34-BAC16) and its revertant (∆ORF34Rev-BAC16), as previously described (20 (link)), were transfected to iSLK cells using Screenfect A plus (Fujifilm-Wako Chemicals) according to the manufacturer’s instructions. Transfected cells were selected under 1000 μg/mL of hygromycin B (Fujifilm-Wako Chemicals) to establish doxycycline-inducible recombinant KSHV-producing cell lines (iSLK-∆34Rev and iSLK-∆34).
To establish stable ORF34-expressing cells for complementation, pCI-blast-3xFLAG-ORF34 and empty vector pCI-blast-3xFLAG were transfected into iSLK-∆34Rev and iSLK-∆34 cells, and transfected cells were selected and maintained in 10 μg/mL and 7.5 μg/mL of Blasticidin S (Fujifilm-Wako Chemicals), respectively. Thus, the stable cell lines iSLK-∆34Rev/pCI-blast-3xFLAG, iSLK-∆34/pCI-blast-3xFLAG, iSLK-∆34/pCI-blast-3xFLAG-ORF34WT, and iSLK-∆34/pCI-blast-3xFLAG-ORF34 mutants were established.
To establish stable ORF34-expressing cells for complementation, pCI-blast-3xFLAG-ORF34 and empty vector pCI-blast-3xFLAG were transfected into iSLK-∆34Rev and iSLK-∆34 cells, and transfected cells were selected and maintained in 10 μg/mL and 7.5 μg/mL of Blasticidin S (Fujifilm-Wako Chemicals), respectively. Thus, the stable cell lines iSLK-∆34Rev/pCI-blast-3xFLAG, iSLK-∆34/pCI-blast-3xFLAG, iSLK-∆34/pCI-blast-3xFLAG-ORF34WT, and iSLK-∆34/pCI-blast-3xFLAG-ORF34 mutants were established.
