Fig. 3.

TLR4 played a crucial role in the M2-CM-induced increase in the malignant properties of HCC cells. a LPS successfully upregulated TLR4 expression in HCC cells. TLR4 expression in HCC cells was detected using western blots and RT-PCR in the M2-CM plus LPS and M2-CM groups. TLR4 upregulation promoted the M2-CM-induced migration of HCC cells. b The distance traveled by migrating HCC cells in the M2-CM + LPS and M2-CM alone groups was measured using a wound-healing assay (× 50). c Cell migration was determined using the transwell assay (× 100). d Analyses of the results of the transwell migration and wound-healing assays. e TLR4 upregulation promoted EMT in cells cultured with M2-CM. The expression of E-cadherin, N-cadherin, and vimentin in the control, M2-CM + LPS, and M2-CM groups was analyzed using western blotting and RT-PCR. f A TLR4 neutralizing antibody inhibited M2-CM-induced HCC cell migration. Cell migration was determined in the M2-CM + TLR4 antibody and M2-CM groups using the transwell assay (× 100). g The TLR4 neutralizing antibody inhibited M2-CM-induced EMT. The expression of E-cadherin, N-cadherin, and vimentin in the M2-CM + TLR4 antibody and M2-CM groups was analyzed using western blots. Data are shown as the means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The data represent at least three independent experiments