Fig. 2: ETI41 and ETI60 inhibit TLR7- and TLR9-dependent downstream signaling.

a, b Western blot analysis was used to quantify the protein levels of phosphorylated p-ERK, p-JNK, p38, Iκ-Bα and p65. RAW 264.7 cells were treated with 5 μM or 10 μM of ETI41 or ETI60, followed by exposure to IMQ (2 μg/ml) for 20 or 30 min (a) and ODN2395 (1 μM) for 40 or 50 min (b) respectively. β-Actin served as a loading control for the cytosol, while lamin A/C was utilized as a loading control for the nucleus. c Protein levels of IRF7 were measured after treatment with 5 μM of ETI41 or ETI60, followed by treatment with ODN2395 (1 μM) for 6 or 8 h. d BMDC cells from ex vivo were used to analyze mRNA expression levels of IL-12p40, IFN-β and CD40. Cells were pretreated with ETI41 (0.2 and 1 μM), followed by treatment with ODN2395 (1 μM) for 3 h. All experiments were independently conducted three times. Statistical differences between the induced and other cases were analyzed and verified using a one-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). e–g The TLR7 agonist IMQ (1 μM) was used in 3’ RNA-seq analysis to induce signaling associated with immune responses in RAW 264.7 cells. RNA-seq analysis showed that ETI41 and ETI60 at a concentration of 10 μM significantly changed gene expression patterns (fold change >2, P < 0.05) in various categories after 2 or 4 h of IMQ treatment. (e–g) depict changes in gene expression categories without pretreatment (e) after pretreatment with ETI41 (f) or after pretreatment with ETI60 (g) followed by IMQ stimulation. h Heatmaps depicting gene expression patterns within the inflammation and immune response categories (fold change >2.5, P < 0.05) of ETI41 or ETI60, dividing the time after IMQ treatment into 0, 2 and 4 h, respectively. Values of each fold change compared with the noninduced case are described, with upregulated genes shown in red and downregulated genes shown in blue.