Fig. 4.

SHIP1 converts PIP₃-rich basolateral membrane into apical domains. a Conversion between the phosphoinositide species, all derived from phosphatidylinositol (PI), occur via the action of kinases (in green) and phosphatases (in red). Dashed arrows, pathway whose occurrence or regulatory enzyme is still unknown. b Heat map of Manhattan-clustered, differentially expressed PIP kinases and phosphatases in cells grown as a monolayer for 48 h or as cysts in Matrigel for 23 or 48 h. Relative mRNA expression levels (log2 values), and clustering categories, are shaded as indicated. Four independent experiments. c SHIP1 localization during lumen formation in MDCK cells expressing SHIP1-EGFP (green) and stained for Podxl (white) and β-catenin or F-actin (red). d Quantitation of cyst phenotypes treated with ethanol control or SHIP1 inhibitor either at the time of plating (d0) and fixed at day 3 (3 day, left), or treated at day 3 (d3) and fixed at day 5 (5 day, right). Values are mean ± s.d. For 3 day, n ≥ 300 cysts assessed from three wells/condition/experiment, four independent experiments, P-values (two-way ANOVA): **P ≤ 0.001, ***P ≤ 0.0001). For 5 day, n ≥ 1900 cysts assessed from three wells/condition/experiment, three independent experiments. e RNA extracts from MDCK cells stably expressing scramble or SHIP1 shRNA were analyzed by RT-qPCR to detect SHIP1 mRNA levels (n = 1 well per condition, from four independent experiments). P-value (Student’s t-test): *P ≤ 0.05. f, g Western blot of WT and phosphatase-dead SHIP1 and GAPDH in total cell lysates of parental (MDCK) or SHIP1-EGFP-expressing cells expressing scramble or SHIP1 shRNA f, and quantitation of cyst phenotypes g. Mean ± s.d., n ≥ 400 cysts from four wells/condition/experiment. P-values: two-way ANOVA): *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001. h PIP₃ [EGFP-PH-Grp1], Podxl and Par3 (left panels) or β-catenin (right panels) localization in cysts expressing scramble or SHIP1 shRNA. Yellow arrowheads, Par3 (left panels) or β-catenin (right panels); blue arrowheads, Podxl. White arrowheads, overlap of apical and basolateral domains. i Immunolabelling of above conditions, with Podxl and β-catenin. Two independent experiments for parental MDCK versus SHIP1-EGFP WT cells and one experiment for parental MDCK cells versus SHIP1-EGFP WT cells or SHIP1-EGFP phosphatase-dead cells. Scale bars, 10 µm