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. 2015 Sep 4;290(42):25595–25608. doi: 10.1074/jbc.M115.661413

FIGURE 6.

FIGURE 6.

Cblb overexpression or HLXB9 knockdown inactivates the oncogenic c-Met pathway. A, overexpression of Cblb or knockdown of HLXB9 decreases c-Met levels. Western blot analysis of the indicated proteins using WCE prepared from MIN6-4N cells transfected (by nucleofection) with empty vector or Cblb expression plasmid, control siRNA (siC), or HLXB9 siRNA (siHB9) and control shRNA (shC) or Nono shRNA (shNono). Cblb overexpression reduced the level of endogenous c-Met. HLXB9 knockdown increased the level of endogenous Cblb and reduced the level of endogenous c-Met. p84 was used as the loading control. The upper band marked pro-c-Met is the glycosylated c-Met precursor form that is cleaved and processed into mature c-Met (lower band) (44). B, reduced cell proliferation from Cblb overexpression but not from HLXB9 or Nono knockdown. An MTT assay assessed cell proliferation of MIN6-4N cells transfected in A. Overexpression of Cblb caused a slight but significant reduction in cell proliferation, but cell proliferation was unaffected upon HLXB9 or Nono knockdown. Note that the Western blots in A were performed using WCE prepared at 96 h post-transfection. Error bar = mean and S.D. from 3 experiments, * = p < 0.05. vec, vector. C, reduced cell migration from Cblb overexpression or HLXB9 knockdown but not from Nono knockdown. Cell migration assay of MIN6-4N cells transfected in A assessed by staining for cells that migrate across a polycarbonate membrane in a Boyden chamber. Stain from the cells was extracted and measured at 560 nm as an index of cell migration. Cblb overexpression or HLXB9 knockdown significantly reduced cell migration. Error bar = mean and S.D. from three experiments. * = p < 0.05. D, soft agar colony formation is unaffected from Cblb overexpression, HLXB9 knockdown, or Nono knockdown. Bright-field microscopy images of colonies (white dots) formed in soft agar by MIN6-4N cells transfected in A. MIN6-4N cells make very small colonies after 4–6 weeks. The number of days to form colonies or the number and size of the colonies was unaffected by Cblb overexpression, HLXB9 knockdown, or Nono knockdown.