Fig. 3.

Effect of the PDPN-neutralizing antibody, NZ-1, on PLT-induced expression of EMT-related genes in TE11A cells. TE11A cells at confluence in 24-well plates were preincubated with NZ-1 or control rat IgG2a (each 2 µg/mL) for 30 min and then treated with platelets (~ 2 × 10⁷/mL) plus EGTA (2 mM) for 18 h. The RNA was extracted, and the expression of the EMT-related genes, vimentin (a), N-cadherin (b), PDPN (c), and PAI-1 (d), was analyzed by real-time PCR. Values are expressed as the mean ± SEM of 4 culture wells from one representative experiment. The platelet-induced increase was normalized by the untreated cells. ***P < 0.001 by one-way ANOVA and Tukey’s test. e Western blot analysis of the effect of NZ-1 on the platelet-induced expression of Vimentin. TE11A cells or original TE11 cells in 12-well plates were treated with platelets without or with rat IgG2a or NZ-1 for 18 h as above. The wells were then supplemented with a twofold volume of fresh medium and cultured for an additional 2 days. f Densitometric analysis of vimentin levels in TE11A cells. The vimentin levels were normalized to platelet treatment alone (none). The data shown are one representative experiment conducted in triplicate cultures and expressed as the mean ± SEM. *P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey’s test