Fig 1.
Generation of BCBL-1 and BC-3 cells with stable HIF-1α knockdown.
(A) Protein levels of HIF-1α in nuclear extracts of BJAB, BCBL-1, and BC-3 cells measured by Western blot analysis after 24 hours in normoxia. Tata-binding protein (TBP) is used as a loading control. BCBL-1 and BC-3 cells were transduced with lentivirus encoding shRNA to HIF-1α or Scrambled (Scr) RNA and stable cell lines were generated with puromycin selection. Total RNA and nuclear protein extracts were extracted from the cells to confirm the status of the knockdown. (B) mRNA levels of HIF-1α measured by RT-qPCR after 48 hours in normoxia(N) or hypoxia(H). mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under normoxia. (C and D) Protein levels of HIF-1α measured by Western blot analysis of nuclear extracts after 24 hours in culture. β-actin is shown as a loading control. (C) Normoxic levels of HIF-1α levels in the absence or presence of 50μM cobalt chloride (CoCl2), a hypoxia mimic that prevents oxygen-induced degradation of HIF-1α. (D) HIF-1α levels under normoxia or hypoxia.
Fig 2.
Effect of HIF-1α knockdown on glycolysis and lipid biogenesis.
(A) Levels of extracellular lactate produced after 24 hours in normoxia (N) or hypoxia (H), expressed as fold change relative to cells containing shScr under normoxia. (B) Protein levels of HIF-1α regulated glycolytic genes after 48 hours in normoxia or hypoxia as assessed by Western blot. β-actin is shown as a loading control. (C) mRNA levels of HIF-1α regulated glycolytic genes measured by RT-qPCR after 48 hours in N or H. mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under N. (D) Levels of miR210 measured by taqman RT-qPCR assay. RNA levels are normalized to that of internal control RNU43 and expressed as fold change relative to shScr cells in normoxia. (E) Levels of phospholipids produced in shHIF-1 cells relative to shScr cells in normoxia. (F) mRNA levels of SIAH2 in BCBL-1 cells normalized to 18S and expressed as fold change relative to shScr cells under N. (G) Protein levels of OGDH2 after 48 hours in N or H. Numbers below the blot for OGDH2 represent protein levels normalized to β-actin and expressed as fold change relative to shScr cells in N. Western blots were done on lysates from three independent experiments and representative blots are shown. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *P ≤0.05, **P ≤ 0.01 (2-tailed t-test, paired).
Fig 3.
HIF-1α knockdown leads to reduced lytic replication of KSHV.
(A) mRNA levels of RTA, vIL-6, and ORF26 (a late lytic gene) of KSHV in BCBL-1 cells measured by RT-qPCR. Error bars represent standard deviation from at least 3 experiments. (B) Western blot showing protein levels of intracellular RTA, vIL6, and β-actin in the cell lysates as well as secreted vIL6 in the supernatant after 48 hours in normoxia (N) or hypoxia (H). (C) Protein levels of ORF45 in concentrated virus particles released in the supernatants after 72 hours. *Unspecific band. Western blots were done on lysates from three independent experiments and representative blots are shown.
Fig 4.
Effect of HIF-1α knockdown on the expression of KSHV latent genes in PELs.
(A) Schematic of multicistronic KSHV latency locus showing the location of latent genes and hypoxia-response elements (HREs) in the promoter region. (B) mRNA levels of latent genes in BCBL-1 cells within and (C) outside of the latency locus as measured by RT-qPCR and normalized to that of 18S RNA. RNA levels for each gene are expressed as fold change relative to shScr cells under normoxia (N). Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *P ≤0.05, **P ≤ 0.01 (2-tailed t-test).
Fig 5.
Effect of HIF-1α knockdown on the expression of KSHV miRNAs.
(A) Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. (B) Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *P ≤0.05 (2-tailed t-test).
Fig 6.
Effect of HIF-1α suppression on growth of PELs.
(A) Growth rate of shScr or shHIF-1 BCBL-1 and BC-3 cells measured by counting live cells every 24 hours in normoxia. (B) Proliferation rate of shScr or shHIF-1 cells measured by MTS assay at 72 hours in normoxia using Promega’s CellTiter 96 Aqueous One Solution assay, which measures the amount of NADH or NADPH produced by metabolically active cells. (C) Colony forming efficiency of shHIF-1 cells relative to shScr cells. Error bars represent standard deviations from at least 3 independent experiments. Results shown in B and C are fold changes compared to shScr cells. Statistically significant differences between shScr and shHIF-1 cells are indicated. *P ≤0.05, **P ≤ 0.01.
Fig 7.
Effects of HIF-1α inhibitor PX-478 on PELs.
(A) HIF-1α mRNA levels in BCBL-1 cells 24 hours after treatment with various concentrations of PX-478, normalized to 18S internal control and expressed as fold changes compared to no PX-478 control cells. (B) Proliferation rate of PEL cell lines BCBL-1, BC-3, JSC-1, BC-1, and BC-2 and Burkitt’s lymphoma (BL) cell lines BJAB and CA46 measured using the MTS assay 72 hours after treatment with indicated concentrations of PX-478, expressed as fold changes compared to no PX-478 control cells. (C) Growth rates of the PEL and BL cells treated with 0 or 10 μM PX-478. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between untreated and inhibitor-treated cells are indicated. *P ≤0.05, **P ≤ 0.01.