Fig. 2.

Validation of PI(3,4)P2 localization. a Schematic representation of a Pipeline for semi-automated phosphoinositide intensity analysis, PAPI. MDCK cysts stably expressing GFP-tagged PIP reporters were cultured in 3D for 48–72 h, fixed and stained with Podxl to mark the apical domain, Phalloidin the cortex, and Hoescht the nucleus. Confocal optical sections of MDCK cysts were imaged and automated processing selected the medial plane based on the maximum lumen area. Separated cyst regions were defined based on differential localization of the above markers. PIP probe intensity was measured in each domain, followed by mathematical and statistical analysis to extract the relative PIP probe intensity ratio within compartments of the same object (cyst). b Cysts at the open lumen stage expressing either EGFP-2xPH-TAPP1 (WT) or a mutant EGFP-2xPH-TAPP1 unable to bind PI(3,4)P₂ (ΔPIP), stained for F-actin (red) and nuclei (blue). Luminal (white arrowheads), basolateral (yellow arrowheads), and nuclear (red arrowheads) localization is highlighted in magnified fields. c Quantitation of relative apical to cytoplasm (left), basolateral to total (center) or nuclear to total (right) PIP reporter intensity compared to GFP-overexpressing control MDCK cells. Box-and-whiskers: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n ≥ 208 cysts assessed from three wells/condition/experiment, three independent experiments (2 for EGFP-2xPH-TAPP1 WT clone 2). P-values (One-way ANOVA): *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0001. d, e Forty-eight hours MDCK cysts stained for endogenous PI(3,4)P₂ (greyscale or green), F-actin (red) and nuclei (blue) using four different fixation and staining protocols, i⁴³, ii¹², iii²⁴, iv⁴⁴. Note that PI(3,4)P₂ can be observed at the luminal domain in all cases (magenta arrowheads). Nuclear localization was also detected in some conditions (iv, e, yellow arrows). f Sequential optical sections of the medial region of an MDCK cyst expressing EGFP-2xPH-TAPP1 (green) stained for endogenous PI(3,4)P₂ (red). Note co-localization in the luminal membrane (white arrowheads) and nuclei. g Inverted polarized MDCK cyst expressing EGFP-2xPH-TAPP1 (green) stained for PI(3,4)P₂ (red) and Hoescht (blue). h Forty-eight hours MDCK cyst expressing a probe for PIP₃ [EGFP-PH-Grp1, green] stained for endogenous PI(3,4)P₂ (red and greyscale) and Hoescht (blue). Yellow arrowheads, basolateral. Magenta arrowheads, luminal. All scale bars, 10 µm