Fig. 7.

INPP4A/B negatively regulate apical polarization. a Analysis of parental MDCK or INPP4B WT or phosphatase-dead (CA, C842A) by western blot, showing INPP4B and β-tubulin expression. b Representative confocal images, cysts stained for Podxl (red), β-catenin (green) and nuclei (blue). mCherry (red). Note defective lumen formation, and subcortical Podxl accumulation upon INPP4B expression, but not the C842A mutant. Scale bars, 10 µm. c Phenotype quantitation without or with overexpression of WT or phosphatase-defective INPP4B. Mean ± s.d., n ≥ 300 cysts assessed from three wells/condition/experiment, three independent experiments. P-values: two-way ANOVA. ***P ≤ 0.0001. d Downregulation of INPP4A/B by shRNA. RNA extracts from MDCK cells stably expressing scramble or shRNA targeting the corresponding gene were analyzed by RT-qPCR. Mean ± s.d., n = three replicate wells/condition, from one experiment. P-values: One-way ANOVA. *P ≤ 0.05. e, f Quantitation f of single lumen formation in 48 h control (EtOH) cysts stably expressing either scramble or INPP4A/B shRNAs, alone or in combination, and repeated in SHIP1-inhibited conditions (SHIP1-i) and representative images of Podxl staining from these condition (e, Podxl in inverted greyscale; scale bars, 50 µm). Values are fold change to control. Plots are box-and-whiskers: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n ≥ 2000 cysts assessed from three replicate wells/condition/experiment, three independent experiments. P-values, Mann–Whitney test: *P ≤ 0.05, **P ≤ 0.005