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. 2020 Nov 26;29(3):1214–1225. doi: 10.1016/j.ymthe.2020.11.026

Figure 2.

Figure 2

Bc-NFκBdODN Inhibits NF-κB Activity in Human and Mouse Target Cells In Vitro

(A) RAW264.7 macrophages stably expressing NF-κB/p65-GFP fusion protein were treated 18 h using 1 μM Bc-NFκBdODN or Bc-scrODN, then stimulated for 30 min with 100 ng/mL LPS. Localization of p65-GFP fusion protein was examined using confocal microscopy (right) and the percentage of cells with nuclear NF-κB signal was quantified (left). Counted were >40 cells/area in triplicate per experimental group. Means ± SD are shown. (B) RAW264.7 macrophages were incubated with fluorescently labeled Bc-NFκBdODNCy3 or CpG-STAT3dODNCy3 (200 nM) for 18 h and then stimulated using LPS (100 ng/mL) for the last 30 min. Cells were fixed, permeabilized, and the interaction between the oligonucleotides and NF-κB was assessed using PLA with Cy3- and NF-κB/p50-specific antibodies or control isotype antibodies followed by secondary DuoLink antibodies with PLA probes. The close proximity of both tested epitopes is indicated by a cyclic polymerase reaction producing green fluorescent spots in the cytosol. (C) Target specificity of Bc-NFκBdODN in cell-free EMSA detecting the effect on the DNA binding by NF-κB (top) or STAT3 (bottom). RAW264.7 cells were stimulated using LPS (100 ng/mL) for 30 min before collecting nuclear extracts, which were then incubated in the presence of 30 nM Bc-NFκBdODN, NF-κB dODN, Bc-scrODN, or CpG-STAT3dODN for 30 min before adding radiolabeled probes for NF-κB (B) or STAT3 (C) and gel shift assays. (D) Bc-NFκBdODN reduces NF-κB activity in lymphoma cells. The A20, U2932, and RaJi cells were treated 18 h using 1 μM Bc-NFκBdODN, Bc-scrODN, or NF-κB dODN, before collection of nuclear extracts and testing by EMSA. (E) Bc-NFκBdODN prevents expression of NF-κB target genes in BCL cells. Human U2932 cells were treated for 3 days using 1 μM Bc-NFκBdODN, Bc-scrODN, or NF-κB dODN, then stimulated with 100 ng/mL LPS for 3 h. The expression of CCND2 and MYC genes was assessed using quantitative real-time PCR; experiments were repeated twice in triplicates. Means ± SEM are shown.