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. 2016 Mar 24;17(5):724–738. doi: 10.15252/embr.201541392

Figure EV7. Usp27x sensitizes 1205Lu melanoma cells to apoptosis induction by inhibition of the Raf‐ERK pathway.

Figure EV7

  1. 1205Lu melanoma cells carrying dox‐inducible GFP‐Usp27x and the same cells where Bim had been targeted using CRISPR/Cas9 (1205Lu‐GFP‐Usp27x/Bim2KO) were treated with dox as indicated. After 48 h, UO126 (10 μM) was added for another 48 h. Apoptosis was measured by staining for active caspase‐3, followed by flow cytometric analysis. All data (means ± SEM) are from n = 5 separate experiments. P‐values (t‐test) for statistically significant differences are shown. The addition of QVD blocked the appearance of active caspase‐3 staining (not shown). Inset shows knockout efficiency of Bim in 1205Lu‐GFP‐Usp27x melanoma cells (n = 2).
  2. GFP‐Usp27x‐ or GFP‐Usp27xC87A‐expressing 1205Lu melanoma cells were treated as in (A) and percentage of cells with active Bax was measured by FACS using a conformation‐specific antibody (6A7 clone) that recognizes only the activated form of Bax. All data (means ± SEM) are from n = 5 (GFP‐Usp27x) or n = 3 (GFP‐Usp27xC87A) separate experiments. P‐values (t‐test) for statistically significant differences are shown.
  3. Dox‐inducible GFP‐Usp27x or GFP‐Usp27xC87A were induced in 1205Lu melanoma cells as indicated. After 48 h, cells were treated with vemurafenib as indicated for additional 24 h. Apoptosis was measured by staining for active caspase‐3 and flow cytometry. Bars show percentage of active caspase‐3‐positive cells from n = 2 independently performed experiments.