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. 2023 Nov 21;14:7353. doi: 10.1038/s41467-023-42994-w

Fig. 6. Expression of Gag caused a reorganization of SM-rich and Chol-rich domains in the PM.

Fig. 6

a Lifetime distribution in the cells labeled with EGFP-NT-Lys (D), EGFP-NT-Lys and mCherry-D4 (DA) in FLIM-FRET. The lifetime τ [ns] in a single-component analysis was shown. b, c Lifetime τ1 [ns] (b) and amplitude α1 [%] (c) of cells doubly labeled with EGFP-NT-Lys and mCherry-D4. b–f DA, WT, WM, ∆L, P99A, and EE indicate cells expressing vectors, Gag WT/Gag WT-mTagBFP2, Gag-WM/Gag WM-mTagBFP2, Gag-∆L/Gag-∆L-mTagBFP2, Gag-P99A/Gag-P99A-mTagBFP2, and Gag-EE/Gag-EE-mTagBFP2, respectively. d Representative images of EGFP-NT-Lys, Gag-mTagBFP2 derivatives, and lifetime τ1 in WT, WM, ∆L, P99A, and EE. τ1 [ns] from 0.8 to 2.2 is indicated in pseudo-colors on the right. Bar, 10 µm. e, f Mean τ1 [ns] (e) or mean α1 [%] (f) in WT, WM, ∆L, P99A, and EE were plotted in every quartile of mTagBFP2 intensity. Pearson correlation coefficients were calculated between mean τ1 or α1 in each range and the rank of the intensity (see “Methods”). The r and p (two-tailed) values were indicated in the graphs. *, **, and *** indicate that there is a correlation at the significance level of 0.05, 0.01, and 0.005. g, h MOC values between AF647-NT-Lys and JF549-D4 in dSTORM imaging of vector (vec) or Gag WT/Gag WT-mEos4b (WT) -transfected cells. g MOCs of AF647-NT-Lys to JF549-D4, h MOCs of JF549-D4 to AF647-NT-Lys. The value in each cell, the mean in each experiment, and the mean ± SEM in the three independent experiments are shown as dots and bars in three different blue colors, and a black bar and error bar, respectively (n = 30 for all except for EE (n = 29) (a–f), and n = 18 and 15 for vec and WT (g and h), over three experiments). The two-tailed paired t-test is applied on (a), (g), and (h), and the one-way ANOVA post-hoc Tukey test on (b) and (c). * and ** indicate significant differences at the significance levels of 0.05 and 0.01, respectively. ns indicates not significant. Source data and detailed statistics were provided as a Source Data file.