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. 2017 Dec 14;20(2):152–164. doi: 10.1016/j.neo.2017.11.007

Figure 1.

Figure 1

Overexpression of Usp9x increases 3D colony formation and invasion of human PANC1 cells.

(A). Immunoblot analysis confirms expression of Flag-tagged Usp9x in human PDAC cell line PANC1. Usp9x-OV increases protein expression levels of Usp9x substrates ITCH and Mcl-1. β-Actin was used as the loading control.

(B) Phase contrast images show effect of Usp9x overexpression on the growth of PANC1 cells in monolayer. PANC1-Usp9x-OV cells show a fibroblast-like phenotype in monolayer cultures as compared to epithelial phenotype of PANC1-vector control cells.

(C) Phase contrast images show effect of Usp9x overexpression on the growth of PANC1 cells in 3D culture on 2% GFR Matrigel. PANC1-Usp9x-OV cells show a typical malignant phenotype with disruption of flat, round 3D colony growth as compared to PANC1-vector control cells. Representative column graph shows an increase in the number of colonies formed in 3D culture in PANC1-Usp9x-OV cells compared to PANC1-vector control cells.

**P < .005.

(D) Phase contrast images show increased spheroid size in suspension culture in PANC1-Usp9x-OV as compared with PANC1-vector control cells. Representative column graph shows an increase in the size of spheroids grown in suspension culture in PANC1-Usp9x-OV cells compared to PANC1-vector control cells. ****P < .00005.

(E) Representative column graph shows increased invasion in PANC1-Usp9x-OV cells through GFR Matrigel invasion chambers as compared to PANC1-vector control cells. *P < .05.