Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT-derived mRNA enhances translation and reduces immune activation. ( A ) Flow cytometry analysis of eGFP expression in HEK293T cells transfected with eGFP mRNA synthesized using either dsDNA or NiLoT, as used in Fig. . Fluorescence intensity was measured at 18 h post-transfection. Cells treated with Lipofectamine TM 3000 alone served as mock controls. Mean fluorescence intensity was normalized to the mock condition. Statistical comparisons were performed using two-tailed, unpaired t -test; ** P < .01 and **** P < .0001. ( B ) Fluorescence microscopy images showing eGFP expression in cells transfected with NiLoT- or dsDNA-derived eGFP mRNA. ( C ) Quantification of IFN-β secretion by ELISA in HEK293T cells transfected with NiLoT- or dsDNA-based eGFP mRNA. Cells treated with Lipofectamine TM 3000 alone served as mock controls, and cells treated with poly(I:C) served as positive controls for immune activation. Statistical comparisons were performed using two-tailed, unpaired t -test; * P < .05, ** P < .01, and *** P < .001. ( D ) Flow cytometry analysis of eGFP expression in HEK293T and THP-1 cells transfected with m 1 Ψ-modified eGFP mRNA synthesized using either dsDNA or NiLoT. Fluorescence intensity was measured at 18 h post-transfection. Cells treated with Lipofectamine 3000 alone served as mock controls. Mean fluorescence intensity was normalized to the mock condition. Statistical comparisons were performed using two-tailed, unpaired t -test; **** P < .0001. ( E ) Fluorescence microscopy images showing eGFP expression in HEK293T and THP-1 cells transfected with NiLoT- or dsDNA-derived eGFP mRNA. ( F ) Quantification of IFN-β secretion by ELISA in HEK293T and THP-1 cells transfected with m 1 Ψ-modified eGFP mRNA synthesized from either NiLoT or dsDNA templates. Cells treated with poly(I:C) were included as positive controls for innate immune activation. ELISA measurements were performed 24 h post-transfection. Statistical comparisons were performed using a two-tailed, unpaired t -test; * P < .05 and ** P < .01.

Article Snippet: After 18 h, 500 μl of supernatant was analyzed using a human IFN-β ELISA Kit (Cusabio, Wuhan, China, #CSB-E09889h).

Techniques: Derivative Assay, Activation Assay, Flow Cytometry, Expressing, Transfection, Synthesized, Fluorescence, Two Tailed Test, Microscopy, Enzyme-linked Immunosorbent Assay, Modification