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

Journal: Oncoimmunology

Article Title: Mild microwave hyperthermia promotes mitotic catastrophe, induces time-delayed cGAS-STING activation and restores sensitivity to anti-PDL1 therapy in Pan02 pancreatic cancer model

doi: 10.1080/2162402X.2025.2602216

Figure Lengend Snippet: (A–I) Representative images of cGAS, STING, pSTAT1, IFNβ, CCL5, CXCL10, HMGB1, PD-L1, and γH2AX immunofluorescent or immunohistochemical stained tissue sections, respectively, of untreated, αPD-L1 mAb-treated, Ht-treated and Ht+αPD-L1 mAb-treated tumors (treatment side and abscopal side).

Article Snippet: The following antibodies were used for western blotting and immunostaining: anti-human cGAS (Cell Signaling), anti-mouse cGAS (SantaCruz), STING (Novus Biological), phospho-IRF3 Ser-386 (Cell Signaling), phospho-IRF3 Ser-396 (Cell Signaling), IRF-3 (SantaCruz), phospho-TBK1 Ser172 (Cell Signaling), TBK1 (SantaCruz), phospho-H2A.X Ser139 (Cell Signaling), PD-L1 (Cell Signaling), CD11c (Cell Signaling), CD8α (Cell Signaling), F4/80 (Proteintech), CD163 (Proteintech), CD86 (Novus Biologicals), FoxP3 (Cell Signaling), Gr1 (Invitrogen), GranzymeB (Invitrogen), Tim3 (Invitrogen), phospho-STAT1 Tyr701 (Cell Signaling), and IFNβ, CCL5, CXCL10, CD56, MICA, HMGB1, SUMO1, SUMO2/3, UBC9, SAE1, UBA2, SENP3, Beta-Actin (all from Proteintech).

Techniques: Immunohistochemical staining, Staining