Supplementary Figure 11: Initial characterization of the StaPLd-Casp9 suicide switch.
From: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
(a) In HeLa Flp-In cells stably expressing catalytically inactive StaPLd-Casp9C287S for 24 h, full-length tandem dimer, revealed by HA blotting, is observed with ASV but not DMSO treatment. However, when cells expressing active StaPLd-Casp9 are treated with ASV, no signal is detected by blotting to HA, β-actin, or coexpressed RFP, suggesting cell loss. Immunoblots are from the same experiment and are subsets of one membrane. Data represent a single experiment. Corroborating results were obtained with wells treated and assayed in parallel (data not shown). (b) Visible rounding and lifting occurs after a 24 h in ASV for cells expressing StaPLd-Casp9 but not StaPLd-Casp9C287S. Scale bar, 500 μm. Data represent a single experiment. Similar results for live StaPLd-Casp9 were obtained in an independent experiment (data not shown). (c) Gating strategy used to process flow cytometry data. Events were first plotted as forward scatter amplitude (FSC-A) vs. side scatter amplitude (SSC-A) to exclude low-scatter debris-like events. Next they were plotted as FSC height (FSC-H) vs. FSC-A to exclude non-singlet events with an inconsistent FSC-H to FSC-A ratio. (d) Flow cytometry of live, annexin-stained stable StaPLd-Casp9-expressing Flp-In HeLa cells reveals extensive apoptosis after 24 h incubation in ASV, as evidenced by annexin signal. HeLa cells stably expressing a catalytically inactive C287S variant serves as a control, and coexpressed RFP reports StaPLd-Casp9 expression. Comparison to parental Flp-In HeLa cells confirms the stable cells were RFP positive. A representative experiment is shown. Comparable results were obtained in a second independent experiment (data not shown).
