Figure EV3. Characterisation of Phosphoinositide enrichment in Invasion assays.

• A, C
  Confocal images, Trp53 ^−/− or Trp53 ^−/−;Pten ^−/− invasive monolayer fronts with cells expressing mNeonGreen (mNG) tagged biosensors for (A) PI(4,5)P₂ (PH‐PLCδ1) or (C) PIP₃ (CYTH3^2G). Representative of (A) 7 (Trp53 ^−/−) or 9 (Trp53 ^−/−;Pten ^−/−) fields or (C) 8 (Trp53 ^−/−) or 9 (Trp53 ^−/−;Pten ^−/−) fields imaged across n = 2 experiments set up with repeated cultures of each subline. Magnified boxed regions, pseudocoloured with FIRE LUT. Arrowheads: cell–cell contacts, black; protrusions, green; cell‐ECM contacts, red. Scale bar, (A)13 μm, (C) 6 μm.
• B, D
  Intensity profiles for mNG PH‐PLCδ1 (B) or mNG PH‐ CYTH3^2G (D) from invasive monolayers on (A, C). Tips measured correspond to boxed, magnified regions on images in (A, C). Arrowhead, phosphoinositide‐rich region.
• E
  Immunofluorescence and confocal imaging of Trp53 ^−/−;Pten ^−/− 1.15 spheroid stained for pS473‐AKT (green or FIRE LUT), F‐actin (magenta or black) and Hoechst (grey). Magnified images from boxed regions. Arrowheads, labelling of pS473‐AKT at protrusion tips. Scale bar, 5 μm. Representative of n = 5 spheroids.
• F
  Intensity profile for pS473‐AKT (green) and F‐Actin (magenta) from spheroid in (A). Tip measured is annotated, ECM to body, yellow arrow; tip, white arrowhead.