Fig. 6.

Class-II PI3-kinases regulate lumen formation. a Downregulation of PIK3C2A, PIK3C2B and PIK3C2G 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 = 3 wells/condition, from one experiment. P-values: One-way ANOVA. *P ≤ 0.05, ***P ≤ 0.0001. b, c Quantitation of single lumen formation in cysts (48 h) stably expressing either scramble or PIK3C2A/B/G shRNAs alone or in combination b and representative images of Podxl staining from these condition (c, Podxl in inverted greyscale; scale bars, 50 µm). Values are fold change to control. Box-and-whiskers: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n ≥ 1900 cysts assessed from three replicate wells/condition/experiment, from two independent experiments. P-values: One-way ANOVA. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005. d–f MDCK cysts stably expressing GFP-PIK3C2A or GFP-PIK3C2B (green), stained for Rab11a (red) at indicated polarity stages. Magenta arrowheads, PIK3C2A/B overlapping with Rab11a vesicles. Yellow arrows, Rab11a-negative, PIK3C2A/B punctate localization. White arrowheads, basolateral PIK3C2B. White arrows, Rab11 positive vesicles. Scale bars, 10 µm. g Quantitation of polarity phenotypes in control (EtOH) or SHIP1 inhibitor-treated (SHIP1-i) parental (MDCK) cysts, or stably expressing GFP-PIK3C2A or GFP-PIK3C2B. Box-and-whiskers: 10–90 percentile; +, mean; midline, median; boundaries, quartiles. PIK3C2A, n ≥ 1550 cysts assessed from three replicate wells/condition/experiment, two independent experiments. P-values, Mann–Whitney test: *P ≤ 0.05, **P ≤ 0.005. PIK3C2B, n ≥ 800 cysts assessed from 2 to 3 replicate wells/condition/experiment, three independent experiments