Figure 1

shRNAs suppress Tat-SF1 expression without cytotoxicity. 1A. Dual luciferase activities were assessed in HeLa cell lysates 48 h post-transfection with shRNA expression cassettes and psiCheck reporter constructs, in triplicate. Target Renilla luciferase levels are given relative to firefly luciferase and normalised to a mock construct with no shRNA expression (U6). shHBVx-5, which targets a sequence in HBV X protein, was included as a negative control. 1B. Total RNA was analysed by qRT-PCR 48 h post-transfection of TZM-bl cells with shRNA expression plasmids, or the U6 mock construct, in triplicate. Tat-SF1 mRNA (htatsf1) levels are given relative to β-actin mRNA (actb) normalised to the U6 control. 1C. TZM-bl cell lysates were subject to PAGE and Western blot 72 h post-transfection. Tat-SF1 expression is given relative to β-actin and normalised to the shHBVx-5 control. 1D. Total TZM-bl RNA isolated 48 h post-transfection was subject to small RNA PAGE and Northern blot. shRNA guide strand expression is given relative to U6 small nuclear RNA and normalised to the U6 control. 1E. TZM-bl cells were stained with Annexin V-conjugated FITC 72 h post-transfection, in duplicate. As a positive control for apoptosis induction, additional cells were treated with 500 nM trichostatin-A (TSA) 16 h pre-stain. Two images were acquired per sample and FITC levels quantified by ImageJ. 1F. Levels of interferon-β mRNA (ifnb1) relative to β-actin mRNA (actb) were determined by qRT-PCR on total cellular RNA extracted 48 h post-transfection, in triplicate. Poly(I:C) dsRNA was used as a positive control. Data are expressed as the mean ± SEM. *, p <0.05, one-way ANOVA with Dunnett post-tests relative to mock construct, U6.