Fig 2: Typical Toll-like receptor (TLR) expression patterns from the human ascending colon. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from ascending colon organ donor (left) and tumor-adjacent normal epithelium (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 20× magnification was used and 50-µm scale bar is in panel I.

Fig 3: Typical Toll-like receptor (TLR) expression patterns from the conventional and germ-free mouse small intestines. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from small intestine conventional (left) and germ-free mice (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 10× magnification was used and 50-µm scale bar is in panel I.

Fig 4: Pacritinib inhibits downstream TLR8-mediated MAP kinase and NF-?B activation. Macrophages were pretreated with pacritinib or controls for 2 h and then treated for 6 h with 5 µg mL-1 GU-rich ssRNA. n = 4. a Right, representative western blots of ERK1/2, JNK, p38, and their phosphorylated forms. Left, densitometric analysis of blots. b Top, representative western blots of Ser32-phosphorylated I?Ba, I?Ba, Ser462-phosphorylated IRF5, and total IRF5 and ACTB. Bottom, densitometric analysis of blots.

Fig 5: GU-rich ssRNAs induce the phosphorylation and ubiquitination of IRAK1 through TLR8. a Macrophages were pretreated with 5 µM MG132, 100 nM zimlovisertib (Zim), or vehicle control (vehicle) for 2 h, then treated for 24 h with 5 µg mL-1 GU-rich ssRNA or controls. Top, representative western blot of IRAK1 and ACTB. Bottom, densitometric analysis of blots. N = 4. b Macrophages were stimulated for 5 h with 5 µg mL-1 RNA40 or RNA649 and IRAK1 immunoprecipitated (IP) from the cell extracts; 30 µL immunoprecipitated IRAK1 was incubated with or without 50 U lambda protein phosphatase (?-PPase) and/or 1.15 µg recombinant human ubiquitin-specific peptidase 2 (USP2) and subjected to SDS/PAGE and western blotting with anti-IRAK1. Phosphorylated IRAK1 and phosphorylated and ubiquitinated IRAK1 are denoted by p-IRAK1 and Ub-IRAK1, respectively. c Macrophages were transfected with TLR8 (siTLR8) or scrambled siRNA (siNS) for 48 h then treated with 5 µg mL-1 GU-rich ssRNA or controls for 24 h. Left, representative western blots of TLR8, IRAK1, and ACTB. Right, densitometric analysis of blots. N = 4.