Measurement for CCCP-induced thermogenesis in brown adipocytes derived from WT-1 cells. (A) Left: Bright-field image of WT-1 adipocytes. Right: Fluorescence image of EuTTA of for the cells on a RT nanosheet. (B) Time course of changes in the EuTTA/Rh101 ratio of RT nanosheets with WT-1 adipocytes upon application of 10 µM CCCP (red) or 0.1% DMSO (black). Values were corrected by those obtained in the medium (see Materials and methods for details). Number of cells, 53 (n = 6) and 38 (n = 5) for CCCP and DMSO, respectively. The horizontal black line indicates the period during which CCCP or DMSO was present. Calibration bar was calculated from the temperature sensitivity of RT nanosheets (−3.35%/°C, cf. Fig. 1 D). Data shown as mean ± SEM. (C) Left: Bright-field image of WT-1 adipocytes on a glass-bottom dish. Middle and right: Fluorescence images of ER thermo yellow and ER tracker green for the cells on left, respectively. (D) Time course of changes in the F.I. of ER thermo yellow (purple) and ER tracker green (green) upon an increase in temperature by IR laser irradiation for 2 s (as indicated by “Heating”). F.I. around the heat center (9,331 µm2) was measured. Data shown as mean ± SEM (n = 4). (E) Time courses of changes in the F.I. of ER thermo yellow in WT-1 adipocytes before and after application of 10 µM CCCP (red) or 0.1% DMSO (black). (F) Same as in E for ER tracker green. In E and F, number of cells, 37 (n = 4) and 43 (n = 4) for CCCP and DMSO, respectively. Data shown as mean ± SEM. (G) Time courses of changes in the F.I. of ER thermo yellow in WT-1 adipocytes preincubated with 10 µM rotenone (blue) or 0.1% DMSO (black) for 30 min and then stimulated by 10 µM CCCP. (H) Same as in G for ER tracker green. In G and H, number of cells, 38 (n = 5) and 29 (n = 3) for rotenone and DMSO, respectively. Data shown as mean ± SEM.
