Fig. 3: Development and utilization of a transgenic C. elegans to detect PTEN signaling.
From: Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
a, Top, representative fluorescence image of labeled C. elegans with pan-neuronal G-PTEN (green) and pharyngeal marker (magenta). Bottom, pseudo-colored FLIM image of the same field of view. Scale bar, 50 μm. b,c, Pseudo-colored FLIM images and quantification in neurons of C. elegans expressing pan-neuronal G-PTEN before (2.63 ± 0.02 ns, n = 81) and after (2.87 ± 0.01 ns, n = 90) 72 h of 500 μM TBB. Scale bar, 20 μm. d, Quantification of fluorescence lifetime of G-PTEN at each stage of larval development, with or without daf-2 RNAi. Larvae were measured at L1 stage (2.51 ± 0.014 ns, n = 14, and 2.64 ± 0.01 ns, n = 44), L2/3 stage (2.57 ± 0.005 ns, n = 184, and 2.63 ± 0.004 ns, n = 204), L4 stage (2.62 ± 0.005 ns, n = 82, and 2.66 ± 0.004 ns, n = 141) or adulthood (P = 0.02, 2.64 ± 0.006 ns, n = 61, and 2.66 ± 0.004 ns, n = 90) without or with daf-2 RNAi, respectively. e–g, Quantification of fluorescence lifetime of G-PTEN transgenic C. elegans neurons comparing each stage of larval development, for control group or with daf-2 RNAi. In e, ***P = 0.0005 and NS P = 0.1004. In f, NS P = 0.7504 and P = 0.9099 for L1 compared to L2/3 and for L4 compared to adult, respectively. ‘n’ denotes number of cells. Error bars represent the s.e.m. Statistical differences for d and e were measured using an unpaired two-tailed student t-test. Statistical differences for f and g were measured using one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. Representative images represent experiments repeated independently at least three times. Significant differences in c–f produced P < 0.0001, unless otherwise stated. ****P < 0.0001.
