Supplementary Figure 1: Additional characterizations in the discovery of FLINC.
From: Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution
(A) Representative images from a single, 35 ms exposure using 561 nm laser on HeLa cells expressing DpTT and control constructs: TagRFP-T alone (TT), co-targeted Dronpa and TagRFP-T (Dp+TT), Dronpa-linker-TagRFP-T (DpTT), EGFP-linker-TagRFP-T (EGTT). Scale bar: 10 μm. (B) Averaged fluorescence intensity of HeLa cells expressing various constructs. n numbers are: TT (8), DpTT (7), Dp+TT (9), EGTT (8), and EGFP-linker-mCherry (EGmCh, n=9). (C) Representative images from a single, 35 ms exposure using 561 nm laser on HeLa cells expressing mutant DpTT constructs: Dp[S142D]-TT (S142D), Dp[C62G/Y63G]-TT (GGG), Dp[N102I]-TT (N102I), Dp[N102I/R149E]-TT (NIRE). Scale bar: 10 μm. (D) Averaged fluorescence intensity of HeLa cells expressing various mutant and control constructs; tandem model DpTT containing wild-type Dronpa (WT), singly-expressed TagRFP-T (TT-alone). n numbers are: TT-alone (8), WT (7), S142D (8), GGG (11), N102I (12), and NIRE (9). (E) Normalized skewness quantification demonstrates that Dronpa fluctuation is not significantly affected by co-transfection (Dp+TT) or fusion (DpTT) with TagRFP-T on the membrane of HeLa cells. (F) Normalized average fluorescence intensity of bacterial colonies in error-prone (K178R, N208K) and site-directed (residues N102 and R149) mutagenesis. Pair-wise t-test results in (B), (D), (E) are marked where data were compared with the construct marked “ref”; n numbers are marked in the corresponding columns. n.s.: not-significant; *: p<0.05; **: p<0.01; ***: p<0.001 where applicable. Center line and whiskers mark mean and s.e.m. values, respectively.
