Fig 1 -

Biological effects of prolonged vIL-6 exposure (A) A schematic diagram of the THP-1 cell culture model. THP-1 cells were treated with or without vIL-6 every other day for 2 weeks (THP-1, vIL-6/THP-1). After withdrawal of the cytokine for 24 hours. vIL-6, hIL-6 and TGF-β were added for 30 minutes for secondary stimulation. Subsequently, 4-Thiouridine (4sU, 300 μM) was added to the culture media and the cells were incubated for 1 hr to label newly synthesized RNA. Newly synthesized RNA was then alkylated by iodoacetamide (IAA, 100mM). Total RNA was isolated for the following analyses. (B) Principal component analysis (PCA). Nascent transcribed gene expression in THP-1 and vIL-6/THP-1 cells was shown. vIL-6, hIL-6 and TGF-β were used as secondary stimulation. Untreated THP-1 cells were used as a control. The samples were represented by three biological replicates. The x and y axes show the percentage of variance explained by PC1 (55% variance) and PC2 (22% variance). (C) The number of upregulated genes (log2 fold change >1, adj p-value < 0.01) after vIL-6 stimulation (left) and TGF-β stimulation (right). Red and green circles represent THP-1 and vIL-6/THP-1 cells, respectively. (D) Comparison of up-regulated gene expression between parent THP-1 and vIL-6/THP-1 cells after stimulation with vIL-6 (left, N = 344) or TGFβ (right, N = 484). Data were analyzed using Wilcoxon matched-pairs signed ranked test and shown as median. (E) KEGG pathway analysis was performed on up-regulated genes (log2 fold change >1, adj p-value < 0.01) in either THP-1 or vIL-6/THP-1 cells with vIL-6 stimulation. Each bar represents the pathways that were enriched only in parental THP-1 cells (red), only in vIL-6/THP-1 cells (green), or commonly enriched (light green). Results are presented in descending order of the analysis. (F) Comparison of all KSHV gene expression between parent THP-1 and vIL-6/THP-1 cells after r219.KSHV infection (N = 88) for 72 hours. Data were analyzed using Wilcoxon matched-pairs signed ranked test and shown as median. (G) RNA-sequence reads aligned to the KSHV genome (NC 009333.1) in parent THP-1 (red) and vIL-6/THP-1 cells (blue) after r219.KSHV infection. (H) KSHV gene expression in parent THP-1 and vIL-6/THP-1 cells after r219.KSHV infection. THP-1 and vIL-6/THP-1 cells were infected with r219.KSHV for 72 hours. RNA was then collected and transcribed into cDNA for RT-qPCR. 18S rRNA expression was used for internal control. Data was analyzed using two-sided unpaired Student’s t test and shown as mean ± SD. (I) KSHV DNA copies 72 hours at r219.KSHV post-infection. GAPDH was used for internal control. Data was analyzed using two-sided unpaired Student’s t test and shown as mean ± SD.