FIG 1.

Increased lytic gene expression following de novo KSHV infection upon dysregulation of KDM2B expression. (A) SLK cells were treated with CoCl₂ for 24 h to induce hypoxia followed by KSHV infection in the presence of CoCl₂ for 72 h. Immunoblot analysis of host (HIF-1α and KDM2B) and viral proteins are shown. HIF-1α induction is an intrinsic marker of hypoxia. The numbers below the KDM2B blots represent the quantification of KDM2B bands in hypoxia samples relative to the normoxia sample (0 μM CoCl₂). (B) Immunoblot analysis of KDM2B expression using anti-FLAG and anti-KDM2B antibodies. (C) SLK cells were transduced with lenti-GFP and lenti-3×FLAG-KDM2B or lenti-shControl and lenti-shKDM2B for 3 days and then infected with KSHV for various time periods. RT-qPCR analysis of RTA expression relative to 18S is shown. (D) Immunoblot analysis of KDM2B expression in the shRNA-treated SLK cells. (E) Viral gene expression measured by RT-qPCR relative to 18S at 72 hpi. (F) Viral DNA level at 72 hpi was measured by qPCR, normalized to the host DNA level, and then calculated relative to 2 hpi in GFP-OE and KDM2-OE cells. (G) EA.hy926 cells were transduced with lenti-GFP or lenti-3×FLAG-KDM2B for 3 days and then infected with KSHV for 3 days. Immunoblot analysis of KDM2B expression using anti-FLAG antibody is shown. (H) Viral gene expression measured by RT-qPCR relative to 18S at 72 hpi. (I) BCBL1 cells were transduced with lenti-GFP or lenti-3×FLAG-KDM2B for 1, 3, or 5 days. 3×FLAG-KDM2B expression was tested by anti-FLAG immunoblotting. Tubulin was used as a loading control. (J) RT-qPCR analysis of viral mRNAs in KDM2B-OE cells relative to GFP-OE cells at 1, 3, and 5 days. t test was performed between GFP-OE and KDM2B-OE or shControl and shKDM2B, and P < 0.05 (*) was considered statistically significant. The numbers on the left side of immunoblots in this and later figures represent the molecular weight in kilodaltons (kDa).