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. 2016 Dec 14;15(8):1452–1462. doi: 10.1038/sj.mt.6300186

Figure 2.

Figure 2

Small interfering RNAs (siRNAs) inhibit efficiently transient replication of reporter hepatitis C virus (HCV) replicon. (a) Graphs represent secreted alkaline phosphatase (SEAP) activities (arbitrary units) in supernatants of En5-3 cell cultures electroporated with no RNA (Mock), 5 μg RNA from HCV Ntat2ANeo replicon (Ntat2ANeo), or a non-replicative HCV RNA (ΔGDD), or coelectroporated with 5 μg of HCV Ntat2ANeo replicon and 2 μg of each indicated siRNA. Each time point corresponds to the amount of SEAP accumulated over 24-hour periods during the first 4 days after transfection, and the last point at day 7 after transfection corresponds to SEAP activity accumulated between days 4 and 7. Each point represents mean ± SD of at least three experiments. Data are represented on a logarithmic scale. (b) Effects of increasing doses of each siRNA (white to dark grey bars for 4–2,000 ng doses) are represented as percentages of SEAP activity inhibition, calculated in proportion to the SEAP activity obtained with replicon Ntat2ANeo RNA alone. Each histogram bar represents mean ± SD of multiple independent experiments. Inhibition doses 50 (ID50s) calculated for each siRNA are indicated on top of the graph. (c) Identical amounts of total RNA extracted from cells transfected with no RNA (Mock), HCV Ntat2ANeo replicon (Ntat2ANeo), or non-replicative HCV RNA (ΔGDD), or coelectroporated with 5 μg of HCV Ntat2ANeo replicon and 2 μg of each indicated siRNA were reverse transcribed and amplified by polymerase chain reaction (PCR) using HCV 5′NTR-specific primers (upper gel) or housekeeping gene GAPDH-specific primers (lower gel). A control reaction performed in the absence of template RNA (No RNA) was analyzed in parallel on 1% agarose gels. PCR products obtained with RNA extracted from two independent cell cultures treated with HCV-specific siRNAs are shown. DNA molecular weight markers are shown on the left of the gels. Filled and open arrowheads point to HCV and GAPDH-specific reverse transcription-PCR products, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NTR, nontranslated region; siIRR, irrelevant siRNA.