Figure 3

PEc overexpression induces EMT in PC-3 cells. (A) Analysis of the morphology of mPC-3 and PC-3PEc cells by light microscopy. PC-3PEc cells have a more spindle-like appearance compared with mPC-3 cells cultured under identical conditions. (B) PC-3PEc cells presented a significant decrease of E-cadherin and a significant increase of vimentin expression compared with mPC-3 cells as assessed by qRT-PCR (Student t test, P < 0.001 for both cases, n = 3). (C) Immunofluorescence (IF) analysis of E-cadherin and vimentin expression in mPC-3 cells and in PC-3PEc cells. E-cadherin expression was suppressed and vimentin was increased in PC-3PEc cells. (D) The treatment of PC-3PEc cells with the anti-human IGF-1Ec antibody (anti-PEc, 1/50) reversed their E-cadherin and vimentin expression profile toward the mPC-3 phenotype (IF analysis). Nonimmunized rabbit serum did not affect E-cadherin and vimentin expression in PC-3PEc cells. (E) Western blot analysis of the Snail, Twist, ZEB1 and cdc6 proteins in mPC-3 and in PC-3PEc cells. These proteins’ expression was induced in PC-3PEc cells. GAPDH and β-actin were used as controls. (F) ZEB1 silencing was associated with an increase in the E-cadherin expression and a decrease in vimentin expression in PC-3PEc cells at 24 h after induction of the silencing, as shown by immunofluorescence. The silencing of the IGF-1R did not affect the E-cadherin and vimentin expression in PC-3PEc cell lines, suggesting that the IGF-1R is not involved in the PEc effects on E-cadherin and vimentin. (G) Analysis of the ZEB1 expression by qRT-PCR in wtPC-3 cells and PC-3 cells after silencing the IGF-1R (PC-3 IGF-1R KD), after the exogenous administration of IGF-1 and PEc (17 nmol/L in both cases). Exogenous IGF-1 induced the ZEB1 expression in wtPC-3 cells 24 h after its administration, but it did not affect the ZEB1 expression in PC-3 IGF-1R KD cells. Exogenous PEc had similar effects on ZEB1 expression that maintained in the PC-3 IGF-1R KD cells. Positive control: human mesenchymal cells (HMC).