Fig. 4.

The activation of TLR2 is essential for inducing DCN⁺ macrophages by B.adolescentis. A The heatmap of differentially expressed TLRs genes in RNA-seq of BMDMs treated with B.adolescentis or vehicle (PBS). B The levels of differentially expressed TLRs genes in BMDMs treated with B.adolescentis were determined by qRT-PCR. C BMDMs were incubated with B.adolescentis or vehicle (PBS) for 24 h. Protein levels of TLR2 and DCN were tested by Western blot. D, E BMDMs were incubated with B.adolescentis or vehicle (PBS) for 24 h with or without 25 μM Cu-CPT22. Protein levels of TLR2 and DCN were tested by Western blot and mRNA level of Dcn was tested by qRT-PCR. F-I THP-1 cells were incubated with B.adolescentis or vehicle (PBS) for 24 h (H-G). THP-1 cells were incubated with B.adolescentis or vehicle (PBS) for 24 h with or without 25 μM Cu-CPT22 (H, I). Protein levels of TLR2 and DCN were tested by Western blot and mRNA level of DCN was tested by qRT-PCR. J-L HCT116 cells were injected into BALB/c nude mice combined with THP-1 cells pretreated with B.adolescentis or vehicle (PBS) for 24 h (n = 5 per group). From the beginning of tumor inoculation until sacrifice, 3 mg/kg Cu-CPT22 or vehicle (5% DMSO) were injected intraperitoneally to mice every two days. Tumor volume was recorded after 6 days. M The positive ratio of Ki67 in mice tumor tissue. The independent experiment was repeated three times. Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; Student t test (B, G), ANOVA test (E, I, K, L, M)