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Figure 4: Signaling pathways underlying the macrophage polarization induced by ectopic endometrial homogenate. THP-1 cell-derived macrophages were treated with either eutopic or ectopic endometrial homogenate or normal endometrial homogenate for 72 h and then subjected to LPS stimulation for 24 h. The mRNA levels of NF-κBp50 (a), Smad2 (b), and Smad3 (c) were detected by RT-PCR. The band intensities (d) were used for quantitative analysis of NF-κBp50 (e), Smad2 (f), and Smad3 (g) protein levels on western blotting. THP-1 cell-derived macrophages were preincubated with NF-κBp50 inhibitor SN50 or Smad2/Smad3 inhibitor SB431542 before addition of homogenates. The mRNA levels of CD86 (h), CD163 (i), IL-12 (j), and IL-10 (k) were detected by RT-PCR. Flow cytometric analysis (l) showed that inhibition of Smad2/Smad3 with SB431542 resulted in a decreased percentage of CD163+ cells in the ectopic group, but not in the eutopic group, in response to LPS stimulation (m). Immunohistochemical staining analysis (n) showed that macrophages treated with ectopic endometrial homogenate had significantly higher IOD values for CD163 staining than those treated with eutopic (o). However, SB431542 could abolish this increasing trend in the ectopic group in the presence of LPS (p). LPS: lipopolysaccharide; SB: SB431542. , , .