Fig 2: Knockdown of WAVE3 negatively affects the survival of pancreatic cancer cells lines. (A and B) Cell growth potential was inhibited in the pancreatic cancer cell lines following transfection with WAVE3 siRNA, as demonstrated by the plate clone formation and CCK-8 assays (*P<0.05 vs. the NC siRNA-trans-fected cells). (C) Flow cytometry demonstrated that the percentages of apoptosis were increased in both cell lines following the knockdown of WAVE3 (*P<0.05) (D) Western blot analyses revealed that the expression of cell survival-related proteins (p53 and Bcl-2) was altered following the knockdown of WAVE3 in the PANC-1 and CFPAC-1 cell lines. *P<0.05 vs. the NC siRNA-transfected cells. WAVE3, Wiskott-Aldrich syndrome protein family verprolin-homologous protein.

Fig 3: Protein level of IGF1R, EGFR and wave3 in MDA-MB-231 and MCF-7 cells with response to the treatment of miR-7 Mimic for 48 h. (A) Predicted 3’-UTR sites in the three genes for miR-7 using online tools. (B) Validation of the miR-7 target sites in Wave3 using luciferase assay. (C–H) Expression of the cellular protein molecules determined using flow cytometry after immunostaining. The primary antibodies used were anti-EGFR (SC-71034. Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IGF1R (SC-712. Santa Cruz Biotechnology), and anti-Wave3 (2806. Cell Signaling Technology), respectively. Isotype antibodies were used as background control. Black histogram: background signal after staining with an isotype primary antibody. Green histogram: Protein expression after treatment with mimic control. Red histogram: protein expression after treatment with miR-7 mimic. Percentages of protein expression was shown after gating against background noise in the overlay plots. (C and D) EGFR expression. (E and F) IGF1R expression. (G and H) Wave3 expression. *P<0.05. A full colour version of this figure is available at the British Journal of Cancer journal online.

Fig 4: WAVE3 is upregulated in ESCC serum. (A) Relative expression of WAVE3 mRNA in serum of patients with ESCC and healthy volunteers. (B) ROC curve of WAVE3 in serum (AUC=0.8325l; P<0.001). ****P<0.0001. WAVE3, Wiskott-Aldrich syndrome verprolin-homologous protein 3; ESCC, esophageal squamous cell carcinoma; ROC, receiver operating curve; AUC, area under the curve.

Fig 5: The PDK2 protein expression levels were altered following WAVE3 siRNA interference. Western blot analysis showed that the knockdown of WAVE3 reduced the expression of PDK2 and the activation of p-AKT 473. *P<0.05 vs. the NC siRNA-transfected cells. PDK2, pyruvate dehydrogenase kinase isoform 2; WAVE3, Wiskott-Aldrich syndrome protein family verprolin-homologous protein 3.