Fig 7. Hsp70 isoforms were not required for RTA-mediated transactivation of KSHV promoters.

(A) HEK-293T cells were transiently transfected with control pEGFP or pRTA-EGFP. 24 h post-transfection immunoprecipitations (IP) were carried out using a GFP-specific antibody. Endogenous Hsc70 interacted with EGFP-RTA but not with the control protein EGFP. (B) HEK-293T cells were transfected with control pEGFP or pRTA-EGFP for 24 h and then analysed by immunofluorescence. Endogenous Hsc70 was cytoplasmic in cells expressing EGFP (i). In contrast, Hsc70 strongly co-localised with EGFP-RTA in the nucleus, but not the nucleoli (ii). (C) HEK-293T cells were transiently transfected with control pEGFP or pRTA-EGFP. 24 h post-transfection, VER-155008 was added and incubated for a further 24 h. Then, immunoprecipitations were carried out using a GFP-specific antibody. Even in the presence of 55 μM VER-155008 the interaction between Hsc70 and EGFP-RTA was not disrupted. (D) HEK-293T cells were co-transfected with pRTA-EGFP along with the Renilla luciferase vector and either the ORF57 promoter firefly luciferase reporter vector, or the empty reporter vector (pGL3-BASIC). The same co-transfections were performed using pEGFP as a negative control protein. 24 h post-transfection, cells were exposed for 2 h to VER-155008 and luciferase activities were measured. Comparable activation of the RTA-responsive ORF57 promoter to that seen in DMSO-treated cells occurred in cells treated with 55 μM VER-155008. The results of three independent transfections were averaged with error bars as standard deviation. (E) HEK-293T cells were transiently co-transfected with pPAN-WT and either pRTA-EGFP or control pEGFP. 24 h post-transfection either vehicle drug DMSO (0.1%) or 45 μM VER-155008 was added and incubated for a further 24 h. Total RNA was then extracted and qRT-PCR performed. RTA-mediated promoter transactivation and subsequent synthesis of PAN RNA occurred at a similar rate in the presence of VER-155008 or control DMSO. The results of three independent transfections were averaged with error bars as standard deviation. (F) The stability of PAN RNA was determined in HEK-293T cells that had been co-transfected with pPAN-WT and pRTA-EGFP. Following 24 h post-transfection, actinomycin D (AcD) (5 μg/ml) or DMSO control (0.5%) was added. Cells were collected over the time points indicated and total RNA was extracted followed by qRT-PCR. The average of two biological replicates with error bar as standard deviation is shown.