Fig 1: HIV-1 Tat and JunB form protein to protein associations and occupy the c-MYC promoter at two AP-1 binding sites. HT1080 cells were transfected with both 500 ng HIV-1 Tat expression vector (pcDNA-Tat) and 500 ng JunB expression vector (pCMV-JunB). (A) The cell lysate was incubated with anti-HIV-1 Tat antibody (ab43014, Abcam, United Kingdom) and pulled down using protein A agarose beads (Roche, Germany). Western blot was carried out using an anti-JunB antibody (Santa Cruz, United States) The goat-anti-rabbit secondary antibody (Bio-Rad, United States) was used as a negative control (lane 3). (B) Diagrammatic representation of the c-MYC promoter depicting the location of the two AP-1 sites in square blocks and the positions of the forward and reverse primers used for the ChIP assay are denoted by black arrows. (C–F) Bar graphs show qRT-PCR data using immunoprecipitated DNA obtained from ChIP with either anti-JunB, anti-HIV-1 Tat or anti-IgG (negative control) antibodies and amplified with the primers depicted in the diagram, error bars represent SD. Statistical significance determined using Student’s t-test in GraphPad PRISM 8, ***(p < 0.001). The graphs are representative of at least three separate repeats.
Fig 2: HIV-1 Tat is detected in BL tumor tissue and elevates c-MYC expression at the transcriptional and translational levels. (A–E) Immunohistochemical detection of Tat protein in FFPE tumor samples from lymphoma patients. An HIV positive BL incubated without primary antibody (replaced with buffer only) (A) and an HIV negative DLBCL incubated with primary Tat antibody (ab43014, Abcam, United Kingdom) as used for the HIV positive BL tumor samples (B), were used as controls. (C) IHC for Tat protein expression in HIV positive BL tumor tissue. Positive staining is indicated by deposition of DAB (brown) and Hematoxylin was used as counterstain (blue/purple). Distinct nuclear Tat expression (marked by black arrows in E). Images (A–C) were taken at 400X magnification and (D,E) at 1000X. Scale bar in (A–C) represents 50 and 20 µm in (D,E). (F) Western blotting of total proteins isolated from Ramos (Left) and BL41 (Right) cells electroporated with pcDNA-Tat expression vector or pcDNA-Empty control using anti-c-MYC (SC-764, Santa Cruz Biotechnology, United States) antibody. Anti-p38 (M0800, Sigma-Aldrich, United States) was used as a loading control. Bar graphs represent densitometric analysis of western blots (ImageJ) and error bars represent standard deviation (SD). (G) Dual-Luciferase Reporter assay shows a significant dose-dependent increase in c-MYC promoter activity with increasing HIV-1 Tat concentration, relative to the empty-vector control which has been set at 1. The following fold increases were obtained for the indicated pcDNA-Tat concentrations: 50 ng: 1.5 ± 0.15; 100 ng: 2 ± 0.03; 200 ng: 2.86 ± 0.2; 400 ng: 3.5 ± 0.2 and 500 ng: 5.3 ± 0.02. Error bars represent SD. (H) Western blot showing increasing expression of HIV- 1 Tat in transfected HT1080 cells in G, with p38 as a loading control. Statistical analysis was done using GraphPad PRISM 8 with Student’s t-test and one-way ANOVA, significance indicates *(p < 0.05), **(p < 0.01) ***(p < 0.001), ****(p < 0.0001). The results are representative of at least three separate repeats.
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