Validation of HIV-1 regulated host cellular factors at the RNA and protein level. (A) Independent validations of randomly selected differentially regulated mRNAs from transcriptome analyses. qRT-PCR was performed to validate the expression of selected mRNAs using specific primer and probe pairs. Fold increase/decrease was calculated based on normalization to RPLPO. Average fold change for each mRNA represents fold change obtained from independent donors (N = 5 per group). (B) Inflammatory factors released by HIV-1 infected PBMC compared to uninfected control cells. Expression of CCL2, CCL8, CXCL5, IL6 and IL8 was monitored by ELISA in supernatants obtained from PBMCs infected with CXCR4-coreceptor utilizing virus (NL43), CCR5-coreceptor utilizing virus (YU2) or mock infected PBMCs (n = 7). * = p < 0.05; NS, Not significant. (C) Expression level of NRGN transcript in PBMCs infected with NL43, YU2 or mock infected cells. Post infection, RNA was isolated and qRT-PCR was performed using NRGN specific primers and probe and results were normalized to RPLPO control. Fold change was calculated using uninfected/mock infected PBMC controls. (D) Immunoblot of NRGN to measure the protein level in PBMC infected with HIV-1 virus or mock. Gag-p24, represents infectivity; and Actin, represents loading control. (E) Densitometry was performed to quantitate the fold change in signal intensity compared to uninfected (NT) and normalized with Actin level. NT, uninfected control; NL4-3, CXCR4 coreceptor virus; and YU2, CCR5 coreceptor virus.
Duskova et al. BMC Infectious Diseases 2013 13:250 doi:10.1186/1471-2334-13-250