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. 2016 May 9;7(31):49143–49155. doi: 10.18632/oncotarget.9237

Figure 1. Efficiency of adenovirus infection and expression of OncomiRs and i-lncRNA.

Figure 1

A. Schematic diagrams of the adenoviruses, the expression cassette of whole-length encoding sequence of i-lncRNA or the EGFP gene was inserted into the EcoRI + SalI sites of adenovirus E1 region, to generate the recombinant adenovirus Ad5F35-i-lncRNA or Ad5F35-EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; mCMV: mouse cytomegalovirus promoter. B. DLBCL cell lines (OCI-Ly10, SUDHL-4, DB) and normal human peripheral B cell line (IM-9) were cultured in 96-well plates at a density of 1 × 104 cells/100 μL/well for 24 h, then infected with Ad5F35-EGFP at MOIs of 10, 50, 100 and 200 pfu/cell. Another 48 h later, the percentages of EGFP-positive cells were observed and counted under a fluorescent microscope; original magnification: 200×. C. The aforementioned cell lines were cultured in 6-well plates at a density of 1 × 105 cells/100 μL/well for 48 h, then harvested for isolation of total RNA, which was used to detect the expression levels of the indicated OncomiRs by qRT-PCR; ***P<0.001. D. Cell lines were cultured in 6-well plates at a density of 1 × 105 cells/100 μL/well for 24 h, then infected with Ad5F35-i-lncRNA at an MOI of 100 pfu/cell. Another 48 h later, cells were harvested for isolation of total RNA, which was used to detect the expression levels of i-lncRNA by qRT-PCR; ***P<0.001 compared with the corresponding parental cells. E. Cell lines were seeded into 24-well plates at a density of 5×105 cells/well and transfected with miRNA inhibitors at a concentration of 100 nmol/L or infected with Ad5F35-i-lncRNA at MOIs of 10 to 200 pfu/cell. After incubation for 24 h, cells were co-transfected with 200 ng/well of pGL3-miRs together with 20 ng/well of pRL-TK using Lipofectamine 2000. At 48 h after transfection, cells were harvested and used to detect the relative luciferase activity with the Dual-Luciferase Reporter Assay, normalized with the activity of pGL3-Control in every cell line; ***P<0.001 compared with the corresponding parental cells.