Extended Data Fig. 5: Application of superPLD to manipulate PA signalling.
From: Activity-based directed evolution of a membrane editor in mammalian cells
a–b, Quantification of nuclear YAP levels to evaluate Hippo signalling activity. HEK 293T cells expressing plasma membrane-targeted optoPLD (PLDdead, PLDWT or superPLDmed) were immunostained for YAP (a; scale bars: 10 µm), and percentage of the total YAP signal that is colocalized with the DAPI (nucleus) signal is plotted for each transfected cell (b). Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 92, 122, and 119, respectively, and n refers to the number of cells examined over three independent experiments). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. ***, p < 0.001. The p values for the indicated pairwise comparisons are <0.0001, <0.0001, and 0.14, respectively. c, Representative Western blots used to quantify p-AMPK levels (see Fig. 5d). Cells expressing plasma membrane-targeted optoPLD were treated with a CaMKK inhibitor (STO-609) for 6 h to block CaMKK-mediated AMPK activation, followed by a 30 min incubation with 488 nm light. d, Representative Western blots used to quantify p-S6K levels (see Fig. 5e). Cells expressing plasma membrane-targeted optoPLD were treated with an AMPK inhibitor (dorsomorphin) for 1 h, followed by a 30 min incubation with 488 nm light. Experiments were repeated at least three times with similar results for c and d.
