Figure 5: Usp9x overexpression in tumours correlates with increased Ets-1 and NRAS protein expression.

(a) Immunoblot for Usp9x, Ets-1, NRAS, pERK and actin in control and Usp9x KD SK-Mel147 cells and HA-Control and HA-Usp9x-overexpressing SK-Mel29 cells. (b) Phase contrast images of SK-Mel29 cells expressing HA-Control or HA-Usp9x and grown on matrigel for 7 days (top) or SK-Mel29 cells expressing Flag-Control or Flag-Ets-1 and grown on matrigel for 7 days (bottom). Scale bars, 100 μm. (c) Phase contrast images of HA-Control and HA-Usp9x expressing SK-Mel29 cells alone or with Ets-1 KD grown on matrigel for 7 days. Scale bars, 100 μm. (d) Colony growth (detected by crystal violet staining) of SK-Mel29 cells expressing HA-Control or HA-Usp9x after 21 days in standard 2D culture (left) or after Ets-1 KD before plating (right). (e) Immunoblot for Usp9x, Ets-1, NRAS and actin in BRAF mutant cell lines 5 days after Usp9x KD. (f) Immunoblot for Usp9x, Ets-1, NRAS, Pan-RAS and actin in NRAS mutant cell lines after 5 days of KD. (g) Phase contrast images of NRAS-mutant SK-Mel2 cells with or without Usp9x KD and Ets-1 KD and grown in 3D (matrigel) for 7 days. Scale bars, 500 μm (100 μm inset). (h) Immunostaining for Usp9x, Ets-1 and NRAS in normal skin, benign nevi, primary melanoma and metastatic melanoma (insets show whole tissue microarray). Scale bars, 20 μm. (i,j) Quantitation of Usp9x, Ets-1 and NRAS immunohistochemical staining by multiplying staining percentage (0–100%) by staining intensity on a numerical scale (none=1, weak=2, moderate=3, strong=4). (k) Immunoblot for Usp9x, Ets-1, NRAS and actin in nine primary and nine metastatic melanoma tumours. (l) Quantification of Usp9x, Ets-1 and NRAS expression in immunoblots from nine primary and nine metastatic melanoma patient tumours.