Fig. 4.
IGFBP3 knockdown impaired EMT program to prevent cellular motility. EPC2–hTERT–EGFR–p53R175H cells expressing shRNA directed against IGFBP3 (BP3-1 and BP3-2) or a scrambled control shRNA (Cont) were subjected to Boyden chamber assays (A) or organotypic 3D culture (B and D). (A) Cells were stimulated with or without TGF-β1 for 14 days prior to the Boyden chamber assays. *P < 0.01 versus Cont plus TGF-β1 (+) (n = 3). (B) Cells were grown in organotypic 3D culture in the absence of exogenous TGF-β. Hematoxylin and eosin staining visualized the reconstituted stratified squamous epithelia and cells displaying downward invasive growth into the stromal compartment. The invasive area, indicated by arrows, was measured and represented as a histogram; magnification, ×200. *P < 0.01 versus Cont (n = 3). (C) Western blotting validates that IGFBP3 expression was restored by ectopic WT or GGG-mutant IGFBP3 in the cells expressing IGFBP3 shRNA. Note that empty vector (Bla) failed to antagonize the IGFBP3 shRNA effect. (D) Cells in (C) were grown in organotypic 3D culture and analyzed as in (B); magnification, ×200. *P < 0.01 versus Cont plus Bla; #P < 0.01 versus BP3-2 plus Bla (n = 3).