Fig 2: Effects of ADAM12 on the epithelial-mesenchymal transition process in a mouse model of peritoneal metastasis. The same number of EV AV construct-, Ssh- or Ash-transfected SW480 cells were injected intraperitoneally into non-obese diabetic/severe combined immunodeficiency mice (n=6/group). The mice injected with the AV-transfected cells showed a significant increase in ADAM12 expression and a decrease in E-cadherin expression compared with in the mice injected with the EV-transfected cells. In addition, the mice injected with the Ash-transfected cells had a significant decrease in ADAM12 expression and an increase in E-cadherin expression compared with in the mice injected with the Ssh-transfected cells. Data are presented as the mean ± SD (n=3). *P<0.05. ADAM12, a disintegrin and metalloprotease 12; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; Ssh, Scrambled pGFP-C-shLenti vector; Ash, ADAM12-pGFP-C-shLenti construct.

Fig 3: Effects of ADAM12 on the EMT process in human colorectal cancer cells. (A) Cell adhesion was measured after AV construct or AS transfection using two cell adhesion substrates, fibronectin and collagen I. The adherent cells underwent the WST-1 assay and adhesion was quantified by measuring the absorbance at 450 nm using a plate reader. (B) Representative western blot images and semi-quantification graphs of EMT-associated proteins including MMP2, MMP9, E-cadherin, Snail, vimentin and claudin-1 in AV-transfected or AS-transfected DLD1 or SW480 cells. (C) Representative western blot images and semi-quantification graphs of basement membrane proteins, including integrin α5, integrin β1 and integrin β3 in AV-transfected or AS-transfected DLD1 or SW480 cells. Data are presented as the mean ± SD (n=3). *P<0.05. ADAM12, a disintegrin and metalloprotease 12; EMT, epithelial-mesenchymal transition; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; SS, scrambled siRNA; AS, ADAM12 siRNA; siRNA, small interfering RNA; MMP, matrix metalloproteinase.

Fig 4: Expression of ADAM12 and E-cadherin in human CRC and lymph node tissues. Immunohistochemistry of human CRC tissues, paired N, ML or NL from the same patients stained for ADAM12 and E-cadherin. (A) Representative images of ADAM12 expression (×200 magnification) in N and CRC tissues. ADAM12 was predominantly expressed in the cytoplasm of CRC cells. (B) Representative images of E-cadherin expression (×200 magnification) in N and CRC tissues. T-WD CRC shows preserved E-cadherin expression and T-PD CRC shows decreased E-cadherin expression. (C) Representative images of ADAM12 expression (×200 magnification) ML. ADAM12 expression in ML was significantly stronger than that in NL. *P<0.05. ADAM12, a disintegrin and metalloprotease 12; CRC, colorectal cancer; N, normal colorectal mucosa; T-WD, well-differentiated tumor; T-PD, poorly differentiated tumor; NL, non-metastatic lymph node tissue; ML, metastatic lymph node tissue.

Fig 5: Effects of ADAM12 on oncogenic signaling pathways in the human colorectal cancer cells. Representative western blot images and semi-quantification graphs for the phosphorylation levels of PDK1, GSK-3β and AKT in AV-transfected or AS-transfected DLD1 or SW480 cells. Data are presented as the mean ± SD (n=3). *P<0.05. ADAM12, a disintegrin and metalloprotease 12; PDK1, phosphoinositide-dependent protein kinase 1; GSK-3β, glycogen synthase kinase-3β; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; SS, scrambled siRNA; AS, ADAM12 siRNA; siRNA, small interfering RNA; p-, phosphorylated; T-, total.