Fig 1: Functional ability of candidate aptamers to inhibit cellular transcriptional response to LT⍺(A) Illustration of signaling pathways induced by LT⍺/TNF⍺-TNFR1. Left: Engagement induces TNFR1 trimerization and recruitment of the adaptor protein TRADD, along with TRAF2, RIPK1, and cellular inhibitors of apoptosis (cIAP1/2), forming the membrane-associated signaling complex I. This complex activates TAK1, which in turn phosphorylates the IKK complex (IKK⍺, IKKβ, and IKKƔ/NEMO). Activated IKK phosphorylates IκB⍺, targeting it for degradation and releasing the canonical NF-κB heterodimer RelA (p65)/p50. RelA/p50 translocate to the nucleus to drive expression of acute inflammatory and survival-associated genes, exemplified here by Cxcl1, Ccl2, and Ccl7. Right: Signaling initiated by binding of LTβR by LT⍺1β2 recruits TRAF2, TRAF3, and cIAP1/2, forming a regulatory complex that controls the stability of NF-κB-inducing kinase (NIK). Ligand engagement leads to TRAF3 degradation and consequent NIK accumulation, enabling activation of an IKK⍺ homodimer independent of IKKβ and NEMO. Activated IKK⍺ phosphorylates p100, promoting its proteolytic processing into p52. The resulting RelB/p52 heterodimer then translocates to the nucleus, driving a distinct transcriptional program associated with lymphoid tissue organization and stromal cell activation, including Cxcl13, Ccl19, and Ccl21, and the transcription of genes involved in lymphoid tissue organization, stromal cell differentiation, and chemokine expression. (B–J) L929 murine fibroblasts were stimulated with LT⍺3 (1 ng/ml) alone or after 1 h preincubation with pateclizumab (PzMAb, 1 μg/ml), LT⍺-specific aptamers (LTa1, LTa5, LTa9, LTa12 at 100 nM), or a scrambled aptamer control (AptScram 100 nM). Temporal induction of cytokine and chemokine transcripts were determined to identify peak induction, while bar graphs show data from a single measured time point (indicated as 1 or 24 h) and the impact of specific inhibitors. Both panels display gene expression changes as mean ± SD ΔΔCt normalized to housekeeping controls. (B–D) (left) Time course and (right) 1-h qPCR analysis of (B) Cxcl1, (C) Ccl7, and (D) Ccl2 expression following LT⍺3 stimulation. (E–G) Expression of non-canonical NF-κB-associated lymphoid chemokine transcripts: (E) Cxcl12, (F) Ccl21, and (G) Ccl19 following LT⍺1β2 stimulation. (Left) Temporal expression kinetics and (right) alterations in 24-h peak cytokine transcript expression levels under the indicated inhibitory conditions. (H and I) (left) Time course and (right) 1-h endpoint expression of (H) Cxcl1, (I) Ccl7, and (J) Ccl2 following TNF-⍺ stimulation in the presence of indicated inhibitors. Statistical significance was determined using two-way ANOVA with Tukey’s multiple-comparison post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.