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Fig. 2 | BMC Infectious Diseases

Fig. 2

From: Inhibition of replication of hepatitis B virus using transcriptional repressors that target the viral DNA

Fig. 2

Assessment of rTALE activity and associated toxicity following transfection of cultured cells with DNA encoding rTALEs. a HBsAg concentrations in supernatants of cultured liver-derived (Huh7) or non-hepatic (HEK293) cells 48 h after transfection with plasmids encoding SPL and SPR rTALEs from the MTTR or CMV promoter/enhancer elements. Control transfections included an irrelevant DNA sequence (Mock, pTZ57R) or plasmids containing a non-HBV targeting rTALE element. b Concentrations of RNA containing HBV core and surface sequences, isolated from transfected Huh7 cells as described in A, were measured using reverse transcriptase quantitative PCR (RT qPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used to normalize the data. c Assay of viral RNA as described in B, but after extraction from HEK293 cells. d Northern blot analysis of RNA extracted from transfected Huh7 cells. Following resolution on formaldehyde agarose gels, blotted RNA was probed with 32P-labeled oligonucleotides complementary to the surface sequence of HBV or human GAPDH. Ratios of bands’ signal intensities following hybridization to HBV and GAPDH probes were calculated after densitometric scanning. Control cells were transfected with a plasmid encoding Green Fluorescent Protein and did not receive HBV sequences. e HBsAg concentrations in supernatants of Huh7 cells 48 h after transfection with plasmids encoding single TALEN subunits (CMV-SPL or CMV-SPR) or pUC118. f Assessment of cell viability using the MTT assay in untransfected liver-derived Huh7 cells and following transfection of cells with indicated plasmids as in A. Data are represented as the means and the error bars indicate the standard errors of the mean. All analyses were carried out on a minimum of 3 replicates and data are represented as the mean ± standard errors of the mean (SEM). Statistically significant differences are indicated by asterisks (**: p < 0.01; ***: p < 0.001)

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