Macrophage migration inhibitory factor is involved in endovascular trophoblast cell function in vitro

Figures

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  Figure 1. MIF specific siRNA reduces MIF expression and cell invasion of HTR-8/SVneo cells. MIF specific siRNA effectively reduced mRNA (a) and protein expression (b, c, d) in whole cell lysates (b, c) and secreted MIF in conditioned media (c, d). Representative Western blots are shown in c. Inhibition of MIF expression had no effect on cell viability (e), but led to a significant decrease in HTR-8/SVneo cell invasion in Matrigel invasion assay (f). Data are presented as mean +SEM, ** p<0.01, *** p<0.001. n = 5 (a), n = 3 (b, f), n=4 (d, e)

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  Figure 2. The effect of MIF knockdown by specific siRNA on mediators of trophoblast invasion in HTR-8/SVneo cells. Integrin subunit α1 was significantly reduced, while subunits α5 and β1 were not changed as assessed by flow cytomerty (a). Gelatine zymography showed a significant decrease in both MMP2 (b) and MMP9 (c) levels. Representative zymogram is shown in d. MFI - mean fluorescent intensity. Data are presented as mean +SEM, * p<0.05, ** p<0.01, *** p<0.001, n =3-6

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  Figure 3. The effect of inhibition of MIF expression on HTR-8/SVneo cell acquisition of endothelial-like phenotype in tube formation assay on Matrigel (a). Total tube length (b) and total number of branching points (c) were significantly reduced. Data are presented as mean +SEM, *** p<0.001. n = 4

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  Figure 4. MIF specific siRNA decreases endothelium replacement capacity of HTR-8/SVneo cells. Inhibition of MIF expression by siRNA (72 h) led to a significant reduction in the capacity of HTR-8/SVneo cells to replace endothelium in trophoblast-HUVEC co-culture model (a, b). Conditioned media from MIF-silenced HTR-8/SVneo cells had no significant effect on HUVEC cell viability in MTT test (c), or adherent cell numbers (crystal violet staining) compared to control. N-cadherin expression was significantly reduced in HTR-8/SVneo cells following MIF knockdown as assessed by flow cytometry (e). MFI - mean fluorescent intensity. Data are presented as mean +SEM, * p<0.05, *** p<0.001. n = 4 (b), n = 3 (c, d)