Fig. 4.

acGM1.8 specifically activates TLR2 signaling. a–c Microarray data on the gene expression in BMDM treated with acGM-1.8 or acGM-0.2: a Venn diagram of the gene numbers, (b) fold change of the markers related to macrophage polarization, and (c) top six signaling pathways changed after the treatment, where the numbers under the circles represent counts of different genes and the toll-like receptor (TLR) signaling pathway shows the greatest change. d, e Detection of secreted embryonic alkaline phosphatase (SEAP) activity in (d) TLR2 and (e) TLR4 reporter macrophages after the treatment of PBS, acGM-1.8, acGM-0.2, or the corresponding agonist. f, g Determination of cytokines (f) TNF-α and (g) IL-12 p70 secreted by macrophages of TLR-knockout mice or wild type mice (*P < 0.05 vs. the PBS group; n = 3). h Detection of acGM-1.8 or acGM-0.2 bound to TLR2: the TLR2 protein was pulled down and the bound polysaccharides were detected by sulfuric-phenol assay (*P < 0.05 versus the group of pure TLR2 without polysaccharides; n = 3). i Measurement of the EC₅₀ value of acGM-1.8: the TLR2 HEK-Blue cells were incubated with acGM-1.8 or Pam₃CSK₄ for 24 h, and the level of SEAP was recorded. j Assessment of potential competition between acGM-1.8 and the classical TLR2 agonist Pam₃CSK₄: the TLR2 HEK-Blue cells were treated with Pam₃CSK₄ alone (1 to 100 ng/mL) or Pam₃CSK₄ (1 to 100 ng/mL) along with acGM-1.8 (fixed at 1 µg/mL), and the level of SEAP was recorded. *P < 0.05 between the two compared groups; k Assessment of the role of TLR1 or TLR6 in acGM-1.8’s activation of TLR2: the TLR2 HEK-Blue cells were pre-incubated with anti-TLR1 or anti-TLR6 before the addition of acGM-1.8 (100 µg/mL). The culture medium was collected and the level of SEAP was recorded. For (k), *P < 0.05 between two inhibition at 100 ng/mL and 1000 ng/mL, n = 3. Data are representative for three independent experiments, except for the microarray data representing two independent biological samples. The original data for (a–c) are submitted to GEO; ns: no significance