**Journal:** The EMBO Journal

**Article Title:** Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

**doi:** 10.1038/s44318-024-00085-6

**Figure Lengend Snippet:** ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

**Article Snippet:** Synthetic lipids diC8 PI (P-0008), diC8 PI(3)P (P-3008), diC8 PI(4)P (P-4008), diC8 PI(5)P (P-5008), diC8 PI(3,4)P 2 (P-3408), diC8 PI(3,5)P 2 (P-3508), diC8 PI(4,5)P 2 (P-4508), diC8 PI(3,4,5)P 3 (P-3908), diC16 PI(4,5)P 2 (P-4516), and diC16 PI(3,4,5)P 3 (P-3916) were purchased from Echelon Bioscience.

**Techniques:** Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy