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. 2024 Jun 6;27(7):110203. doi: 10.1016/j.isci.2024.110203

Figure 3.

Figure 3

Phospomimetic DLC3 PBR mutants show impaired membrane association in cellulo

(A) Localization of GFP-DLC3 K725E or phosphodeficient S208/215A or phosphomimetic S208/215D muteins in transiently transfected MCF7 cells. GFP- and E-cadherin-specific immunostainings plus nuclear counterstain (DAPI). Images are maximum intensity projections of several confocal sections. Scale bars: 20 μm.

(B) Graph shows the mean fluorescence intensity (MFI ±SEM) of the GFP signal at cell junctions versus the cytoplasmic GFP signal (n = 3, N = 39, 33, 30); one-way ANOVA with Dunnett’s post-test: WT vs. AA p = 0.8765; WT vs. DD p = 0.0037).

(C and D) Fluorescence recovery [%] after photobleaching cell-cell contact regions of transiently transfected MCF7 cells expressing GFP-DLC3 K725E with wild-type PBR (WT) or phosphomimetic S208/215D mutations (DD). Images show an exemplary site immediately before (−2 s), immediately after (bleach) and 60 s after photobleaching. Red outline indicates photobleached region. Graph shows mean ± SD, N = 9, 11 from two independent experiments. Intensity curves were analyzed by one-phase association nonlinear regression to obtain half-time of fluorescence recovery (thalf) and mobile fraction (plateau) (t test thalf: p = 0.0445; t test plateau: p = 0.1485; shown is mean ± SEM).