Figure 4
Attenuation of shRNA-mediated Tat-SF1 suppression over time. Samples were isolated from SupT1 cells with stable shRNA expression at time points equivalent to those of the HIV-1p81A-4 replication assay. 4A. Total SupT1 RNA was analysed by qRT-PCR, in triplicate. Target mRNA levels are given relative to β-actin mRNA (actb) normalised to the U6 cell line. Left panel: Tat-SF1 mRNA (htatsf1). Right panel: LEDGF/p75 mRNA (psip1). 4B. SupT1 cell lysates were subject to PAGE and Western blot. Day 20 samples were prepared in duplicate and representative blots are shown. Mean Tat-SF1 expression is given relative to β-actin and normalised to the U6 control at each time point. 4C. Nuclei isolated from SupT1 cells were subject to nuclear run-on analysis to quantify htatsf1 transcription, in triplicate. Samples from both shLTR-U5- and shhtatsf1-a-expressing cells were normalised to those isolated at a time point equivalent to day 0 of the HIV-1p81A-4 replication assay. 4D. Total SupT1 RNA was subject to small RNA PAGE and Northern blot to assess shhtatsf1-a guide strand expression relative to 5S rRNAs. 4E. Proportion of GFP+ SupT1 cells. SupT1 cell populations were analysed by flow cytometry with 5 × 103 events acquired per sample. 4F. shRNA-expressing SupT1 cell lines were cultured for 20 days prior to quantification of cellular DNA. Data are expressed as the mean ± SEM. *, p <0.05, two-way ANOVA with Bonferroni post-tests.