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. 2015 Oct 12;6(35):37792–37807. doi: 10.18632/oncotarget.6096

Figure 3. CrkY251 phosphorylation drives Abl transactivation and regulates distinct biological phenotypes in GBM cell lines.

Figure 3

A. Upon stimulation with hEGF, Crk and Abl kinase are phosphorylated within 1min in U138 and HS683 GBM cells. B. Canonical Crk signaling is seen by CrkY221 phosphorylation by hEGF in GBM cell lines. C. Schematic figure of Crk indicating Tyr 221, 239 and 251 in context of its modular domains. D. Parental HS683 cells were pretreated with or without 10uM Imatinib and stimulated with or without hEGF after which detergent lysates were made and immunoblotted with Abl pY245 and Crk pY251 antibodies. E. Western blot analysis performed to assess the stable expression of MSCV-EYFP (Empty vector), MSCV-EYFP-Crk and MSCV-EYFP-CrkY251F in HS683 cells. F. Wound healing assay: Cell migration of the HS683 stable cells was assessed by imaging the rate of wound healing by serum starved, hEGF stimulated cells in triplicate samples at 6 and 12 hours time points. G. Percentage wound healing was calculated in all cell lines and data were represented as mean ± SEM (n = 3) (P < 0.05). H. Cell invasion by Matrigel-coated Boyden chambers: Post-serum starvations for 12-16hrs, 10,000 cells were seeded in serum free media. 10% FBS containing media was used as chemoattractant in the lower chamber. Cell were fixed, stained and imaged as described (see methods) and quantified to represent data in fold change in cell invasion (left panel). I. Cell invasion using real-time xCELLigence-based assay: Overnight serum starved cells were seeded in serum free media in triplicates in 40000 cells/well. Cell invasion was assayed every 15min for indicated time using 10% FBS containing media as chemoattractant (P < 0.05). J. xCELLigence assay to test the effect of Imatinib treatment on cell invasion of Hs683 cells. K. Distinct morphological features by cells expressing vector (EYFP), Crk, or CrkY251F mutant. L. Micrographs of cell spreading on fibronectin coated dishes: 1000 respective cells were seeded on fibronectin pre-coated dishes (10ug/ml) and images were taken from multiple fields (3 fields/cell line shown) 30min later using 40X objective of phase contrast microscope. M. Real-time xCELLigence-based cell spreading assay on fibronectin-coated CIM-16 plates seeded with 10,000 vector, Crk, CrkY251F expressing HS683 cells using 10%FBS as chemoattractant. N. CrkY251 phosphorylation by fibronectin in stable cell lines: The stable cell lines that were seeded on fibronectin dishes for 30min were harvested, lysed and immunoblotted for endogenous and EYFP- CrkY251 phosphorylation status. O. Tabular summary of the CrkY251-mediated biological phenotypes observed. See also Supplementary Figure S3, S4 and S5.