Fig 2: The effect of HZ08 on phosphorylation of PI3K, Akt and IKKα. a and b, PC-3 cells were treated with HZ08 alone (a) or treated with HZ08 and IR (b). Cellular extracts from the treated cells were used to quantify the levels of PI3K, p-PI3K, Akt, p-Akt, IKKα, p-IKKα by Western blots. c and d, PC-3 cells were pretreated with LY294002 (25 μM), HZ08 (5 μM) and treated with 6 Gy IR. P-Akt, MnSOD in cellular extracts and RelB in nuclear extracts were quantified by Western blots (c). Cytotoxicity was analyzed using MTT by three independent experiments and plotted as mean ± SD. ** (P < 0.01) shows the significances between two groups as indicated (d)

Fig 3: Model of HIV-1 transcriptional activation by Tat and RelB. HIV-1 Tat interacts with RelB. RelB and Tat are recruited to LTR DNA to assist transcription, which increases recruitment of RNA Pol II to the LTR and leads to greater expression of viral genes

Fig 4: Enhanced nuclear staining of RelB in OVCAR8-NUAK1KO xenograft tumours. (A) Representative immunohistochemistry DAB channel images for RelA and RelB in xenograft tumours using OVCAR8 and OVCAR8-NUAK1KO cells. Scale bars represent 100 μm. (B, C) Quantification of DAB staining intensity for RelA (B) and RelB (C) as determined by the IHC Profiler ImageJ plugin. Data are shown as the percentage of pixels classified as “Negative”, “Low Positive”, “Positive”, and “High Positive” for the full image (Total) or nuclear regions as defined by hematoxylin staining (Nuclear). OVCAR8-NUAK1KO were compared with OVCAR8 by unpaired, two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n ≥ 10 tumour nodules per group). Error bars indicate standard error of the mean (S.E.M.).

Fig 5: RelB increases HIV-1 gene transcription. a HEK 293T cells (0.5 × 106) in a 6-well plate were transfected with increasing amounts of pHA-RelB, together with pVSV-G (0.3 μg) and pNL4-3.Luc.env−. (1 μg). Forty-eight hours post transfection, cells were collected for western blotting and luciferase activity analysis. b Stable RelB-overexpressing cells, RELB−/− cells, and control HEK 293T cell lines (0.5 × 106) were transfected with pVSV-G (0.3 μg) and pNL4-3.Luc.env− (1 μg). Western blotting and Luciferase activity essays were performed to assess viral gene expression. c Control and RELB−/− HeLa cells were transfected with pHA-RelB (500 ng) or vector DNA. Twenty-four hours post transfection, cells were infected with VSVG-NL4-3.Luc.env− equivalent to 20 ng p24 for 40 h. Cells were collected for luciferase assay and western blotting analyses. d and e RelB overexpressing or RELB−/− Jurkat cells were infected with VSVG-NLENY1-ES-IRES equivalent to 20 ng p24 for 48 h. Gating on live Jurkat cells was based on size (FSC-H) and granularity (SSC-H). YFP positive cells were scored by flow cytometry 48 h after infection (d). Results from three independent experiments were summarized, and cells were collected for western blotting analysis (e). f PBMCs (2 × 106) were infected with RelB-expressing VLPs, or transfected with siRNA. After 24 h, infection with VSV-G pseudotyped NL4-3.Luc.env− (equivalent to 500 ng p24) was performed by spinoculation at 300×g for 30 min. After 24 h, whole cell lysates were examined in luciferase assay and western blotting with the indicated antibodies