Temperature measurement for HEK293 cells expressing malignant hyperthermia-causing RYR1 mutants. (A) Illustration showing that the expression of mutant RYR1 in HEK293 cells causing the leak of Ca²⁺ from the ER. The magnitude of Ca²⁺ leak is greater for Y523S than R164C (Murayama et al., 2015). (B) Fluorescence images of HEK293 cells on RT nanosheets with or without RYR1 expression. RYR1 was visualized by immunostaining. From left to right: control HEK293 cells, HEK293 cells expressing WT RYR1, HEK293 cells expressing RYR1 (R164C), and HEK293 cells expressing RYR1 (Y523S). (C) Top: Bright-field images of various types of HEK293 cells on RT nanosheets. Middle: Normalized EuTTA fluorescence images of RT nanosheets (see Fig. S6). Bottom: EuTTA/Rh101 images of RT nanosheets. From left to right: control HEK293 cells, HEK293 cells expressing WT RYR1, HEK293 cells expressing RYR1 (R164C), and HEK293 cells expressing RYR1 (Y523S). (D) Comparison of normalized F.I. for EuTTA (left), Rh101 (middle), and the EuTTA/Rh101 ratio (right) on RT nanosheets. Number of cells, 43, 38, 31, and 42 for control (Ctrl) HEK293 cells, HEK293 cells expressing WT RYR1, RYR1 (R164C), and RYR1 (Y523S), respectively. The Steel–Dwass test was performed. a.u., arbitrary units; n.s., not significant.