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Metabolic reprogramming of Kaposi’s sarcoma associated herpes virus infected B-cells in hypoxia

Fig 2

Differential expression of KSHV encoded gene expression in CoCl2/1%O2 induced hypoxia.

BJAB-KSHV cells were grown under normoxic or CoCl2 induced hypoxic environment for 48 hours followed by RNA isolation. The duplicate RNA samples were prepared for sequencing using Illumina RNA sequencing sample prep kits. The indexed ready to run samples were run on Illumina platform at the University of Washington (Core services). The reads alignment with KSHV genome, fold change differences and statistical analysis in terms of p-value were calculated by CLC bio software (Qiagen Inc., Hilden, Germany). The differences in fold change were represented by heat maps using Partek software. (A) Histogram showing transcript coverage for KSHV encoded genes. (B) Heat map for fold change expression of KSHV-encoded genes based on analysis of RNA sequencing data. Asterisk (*) denotes statistical significance with p<0.05; (n.s denotes non-significant difference). (C) Validation of RNA sequencing data by real time PCR in CoCl2 induced hypoxia. BJAB-KSHV cells were grown under normoxic or CoCl2 induced hypoxia followed by RNA isolation and real time PCR. Histogram represents real time PCR fold change of reverse transcribed KSHV encoded genes transcript to cDNA of BJAB-KSHV cells grown in CoCl2 induced hypoxia. Asterisk (*) denotes statistical significance with p<0.05. (D) Validation of RNA sequencing data by real time PCR in 1% O2 induced hypoxia. BJAB-KSHV cells were grown under normoxic or 1% O2 induced hypoxia followed by RNA isolation, conversion to cDNA and real time PCR. Histogram represents real time PCR fold change of KSHV encoded transcripts converted to cDNA in BJAB-KSHV cells growing in 1% O2 induced hypoxia. Asterisk (*) denotes statistical significance with p<0.05; (n.s denotes non-significant difference). (E & F) Fold change expression of KSHV encoded vGPCR, K1, vFLIP, LANA, vCyclin and RTA in KSHV positive BC3 cells grown under normoxia and CoCl2/1% O2 induced hypoxia. BC3 cells were grown under normoxia or CoCl2 induced hypoxia (48 hours) or 1% O2 (24 hours) followed by RNA isolation and cDNA synthesis. Real-time PCR assays were performed using gene specific primers. All the experiments were performed at least in triplicate and the statistical significance in the terms of p-value were determined using graph pad software. Asterisk (*) denotes statistical significance with p<0.05.

Fig 2

doi: https://doi.org/10.1371/journal.ppat.1007062.g002