Figures & data
Fig. 1. In vitro characterization of FLIPSuc90µ∆1Venus for trehalose.
Notes: The dependency of trehalose concentration is represented as a Venus/CFP ratio. The fitting titration curve to a single-site binding isotherm is described, and Values and error bars show means ± SEM (n = 3).
Fig. 2. Intracellular trehalose monitoring by FLIPSuc90µ∆1Venus.
Notes: The intracellular trehalose was observed using HEK293T cells co-expressing FLIPSuc90µ∆1Venus and PvTRET1 at 48 h after transfection. (A) The cytoplasmic fluorescent ratio is shown as Venus/CFP ratio, HEK293T cells co-expressing FLIPSuc90µ∆1Venus and PvTRET1 (magenta), and expressing FLIPSuc90µ∆1Venus (black), respectively. The temporal fluorescent ratio was monitored during perfusion with HBSS buffer. Arrowheads show a time-point in 40 mM trehalose. Values and error bars represent means ± SEM (n = 8). (B) The visualization of trehalose by the fluorescence of the FRET sensor. The fluorescence under the trehalose-free condition is shown (left), and that under the buffer containing 40 mM trehalose (right). Each inset: Cytoplasmic Venus/CFP ratio. The time-lapsed ratio movie is shown as the supplemental movie 1.
Fig. 3. Sensitivity of FLIPSuc90µ∆1Venus in cytoplasm.
Notes: (A) The cytoplasmic fluorescent ratio is shown as Venus/CFP ratio in HEK293T cells co-expressing FLIPSuc90µ∆1Venus and PvTRET1 (magenta), and expressing FLIPSuc90µ∆1Venus as a control, respectively. Each arrowhead represents at a time-point perfused the HBSS buffers in various trehalose concentrations. The error bars showed ± SEM (n = 8). (B) Quantification of intracellular trehalose in HEK293T cells. [Trehalose]i means the concentration in a cell. Values and error bars indicate means ± SEM (n = 3).
Supplemental material
