Fig. 5: PAI-1 induces the migration and invasion of macrophages by activating Akt and Erk1/2 signaling pathways via LRP1. | Laboratory Investigation

Fig. 5: PAI-1 induces the migration and invasion of macrophages by activating Akt and Erk1/2 signaling pathways via LRP1.

From: PAI-1 derived from cancer-associated fibroblasts in esophageal squamous cell carcinoma promotes the invasion of cancer cells and the migration of macrophages

Fig. 5

a Transwell assay of the effect of CAF9 cell coculturing on the migration of macrophages. b Transwell assay of the effect of CAF9 cell coculturing on the invasion of macrophages. c Transwell assay of the effect of a PAI-1 neutralizing antibody on the migration of macrophages cocultured with CAF9 cells. Normal goat IgG was used as negative control for the PAI-1 antibody. d Transwell assay of the effect of PAI-1 neutralizing antibody on the invasion of macrophages cocultured with CAF9. Normal goat IgG was used as negative control for the PAI-1 antibody. e The levels of LRP1 mRNA in macrophages by RT-PCR using GAPDH as the control. f The expression level of LRP1 protein in macrophages by western blotting. Anti-LRP1 and β-actin antibodies were used. g Transwell assay of the effect of rhPAI-1 on the migration of macrophages. h Transwell assay of the effect of rhPAI-1 on the invasion of macrophages. i Representative western blots of the effect of rhPAI-1 on the phosphorylation levels of Akt and Erk1/2 in macrophages. Macrophages in serum-free conditions were treated with 10 ng/mL rhPAI-1 for 0, 10, 30, and 60 min. Western blotting was conducted with total protein extracted from macrophages using specific antibodies against Akt, p-Akt (Ser473), p-Akt (Thr308), Erk1/2, p-Erk1/2 (Thr202/ Tyr204), and β-actin. j Transwell migration assay of macrophages with or without 10 ng/mL rhPAI-1 combined with a PI3K inhibitor (LY294002, 10 μM) or a MEK1/2 inhibitor (PD98059, 10 μM). k Transwell invasion assay of macrophages with or without 10 ng/mL rhPAI-1 combined with a PI3K inhibitor (LY294002, 10 μM) or a MEK1/2 inhibitor (PD98059, 10 μM). l Macrophages were transfected with 20 nM siRNA targeting LRP1 (siLRP1). siNC was transfected into macrophages as a negative control. LRP1 knockdown was confirmed by RT-PCR using GAPDH as control. m Effective knockdown of LRP1 was confirmed by western blotting using antibodies against LRP1 and β-actin. n Transwell assay of the effects of LRP1 knockdown on the migration of macrophages with or without 10 ng/mL rhPAI-1. o Transwell assay of the effects of LRP1 knockdown on the invasion of macrophages with or without 10 ng/mL rhPAI-1. p Western blotting was performed on the total protein of LRP1-silenced macrophages using specific antibodies against Akt, p-Akt (Ser473), p-Akt (Thr308), Erk1/2, p-Erk1/2 (Thr202/ Tyr204), and β-actin compared to the control. For (a–d), (g), (h), (j), (k), (n), and (o), migrating and invading cells were counted in four randomly chosen fields, and data represent the mean ± SEM of triplicate wells for three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).

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