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

Figure 3. Usp27x expression sensitizes 293FT cells to apoptosis induction by stimulation with PMA .

Figure 3

  1. 293FT‐TetR‐3xFlag‐Usp27x or 3xFlag‐Usp27xC87A, a polyclonal derivative where the Bim locus had been targeted by CRISPR/Cas9 and four single clones obtained by serial dilution from the polyclonal line, was treated as indicated (PMA 16.2 nM, QVD 10 μM). Apoptosis was measured after 24 h of treatment by staining for active caspase‐3, followed by flow cytometric analysis. Data (means ± SEM) are from n = 14 (3xFlag‐Usp27x line and 3xFlag‐Usp27xBim2KO polyclonal line), n = 5 (3xFlag‐Usp27xC87A and 3xFlag‐Usp27xBim2KO clone #14), n = 7 (3xFlag‐Usp27xBim2KO clone #11) or n = 4 (3xFlag‐Usp27xBim2KO clone #4 and #7) separate experiments. P‐values (t‐test) for statistically significant differences are shown.
  2. The same cells as in (A) (except Usp27xC87A mutant) were treated as in (A) and stained for active Bax. Data (means ± SEM) are from n = 8 (3xFlag‐Usp27x line and 3xFlag‐Usp27xBim2KO polyclonal line), n = 5 (3xFlag‐Usp27xBim2KO clone #11 and #14) or n = 4 (3xFlag‐Usp27xBim2KO clone #4 and #7) separate experiments. P‐values (t‐test) for statistically significant differences are shown. Expression levels of Bim, β‐TrCP and Flag‐Usp27x of the different cell lines are shown in Fig EV5E.