Figure 1: Effect of Usp9x KD and DUB inhibitor (G9) on the growth and expansion of melanoma cells. | Nature Communications

Figure 1: Effect of Usp9x KD and DUB inhibitor (G9) on the growth and expansion of melanoma cells.

From: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma

Figure 1

(a) Immunoblot for the protein indicated in control and Usp9x KD (shRNA) melanoma cell lines. (b) Phase contrast images of BRAF mutant cells with or without Usp9x KD, grown in monolayer (2D—top) and matrigel (3D—bottom panels) for 7 days. Scale bars, 500 μm. (c) Phase contrast images of NRAS mutant cells with or without Usp9x KD, grown in 2D and 3D. Scale bars, 500 μm. (d) Quantification of colony growth in BRAF and NRAS mutant cells with and without Usp9x KD 7 days after plating. (e) Cell growth (by MTT) of NRAS mutant (SK-Mel2, WM1366, SK-Mel147, SK-Mel103) and BRAF mutant (SK-Mel94, SK-Mel29) cells treated with G9 at the indicated concentrations. The chemical structure of G9 (EOAI3401243) is shown. (f) DUB activity by HA-UbVS labelling in NRAS-mutant melanoma cells grown in 2D (monolayer) or 3D (agarose) and treated with G9 (5 μM, 4 h); HA-UbVS-labeled Usp9x is noted (top); Usp9x protein levels (bottom). (g) Phase contrast images of SK-Mel2 melanoma cells on agarose treated with or without 1 μM G9 for 3 days (left), and phase contrast images of control or Usp9x KD SK-Mel2 melanoma cells grown on agarose 3 days (right). (h) Phase contrast images of NRAS mutant (SK-Mel147) and BRAF mutant (A375) melanoma cells treated with G9 on matrigel for 3 days (left) and phase contrast images of NRAS mutant (SK-Mel103) melanoma cells treated with low dose of G9 (0–1 μM) on matrigel for 3 (left) or 10 days (right). Scale bars, 100 μm.

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