Extended Data Fig. 6: In vitro characterization of the PTEN sensor.
From: Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
a Representative images of fluorescent intensity in HEK293 cells expressing either mEGFP-PTEN-sREACh WT, and with the R14G or the K13E mutation. Scale bar; 20 μm. b Quantification of normalized intensity of Cytosol / Nucleus in HEK293 cells expressing either mEGFP-PTEN-sREACh WT (1 ± 0.05, n = 137), with the R14G (ns, 1.05 ± 0.04, n = 143) or K13E mutation (****,0.16 ± 0.003, n = 209). ns p = 0.5206. c Quantification of fluorescent lifetime in HEK293 expressing G-PTEN before pharmacology application (2.22 ± 0.004 ns, n = 75), after overnight application of 10 μM MG132 (****, 2.32 ± 0.004 ns, n = 117), after 3 hr of 50 μM TBB application (****, 2.38 ± 0.003 ns, n = 50), and after 3 hr of 50 μM TBB application following overnight application of 10 μM MG132 (****, 2.44 ± 0.004 ns, n = 120). ns p = 0.088 for R14G + TBB vs. R14G + MG132 + TBB. d Quantification of fluorescent lifetime in HEK293 expressing mEGFP-PTEN-sREACh 4 A before pharmacology application (2.50 ± 0.003 ns, n = 60), after overnight application of 10 μM MG132 (***, 2.48 ± 0.004 ns, n = 126), after 3 hr of 50 μM TBB application (ns, 2.49 ± 0.002 ns, n = 50), and after 3 hr of 50 μM TBB application following overnight application of 10 μM MG132 (ns, 2.47 ± 0.003 ns, n = 108). *** p = 0.0004 for 4 A vs. 4 A + MG132. ns p = 0.5678, p = 0.0654 and p = 0.9890 for 4 A vs. 4 A + TBB, 4 A + TBB vs. 4 A + MG132 + TBB, and 4 A + MG132 vs. 4 A + MG132 + TBB respectively. e Quantification of dimerization, measured by fluorescent lifetime in HEK293 expressing PTEN-sREACh WT and co-expressing either mEGFP-PTEN WT (****, 2.69 ± 0.001 ns, n = 144 and 2.67 ± 0.001 ns, n = 140), mEGFP-PTEN R14G (****, 2.69 ± 0.001 ns, n = 140 and 2.67 ± 0.001 ns, n = 130) or mEGFP-PTEN C124S (****, 2.66 ± 0.001 ns, n = 161 and 2.62 ± 0.001 ns, n = 244), before and after 3 hr of 50 μM TBB application respectively. ‘n’ denotes number of cells, Error bars represent s.e.m. statistical differences for (b, c, d, e) were measured using one-way ANOVA followed by post-hoc Tukey’s multiple comparison. Representative images represent experiments repeated independently at least 3 times. Significant differences in (b-e) produced p < 0.0001, unless otherwise stated in the legend above. **** p < 0.0001.
