Fig. 2: Manipulation of intraluminal pressure does not significantly affect lumen shape.

a Representative cross-sections of MDCK spheroids grown for 3 days imaged using differential interference contrast treated with vehicle (left), ddAVP (center), or ouabain (right) for 4 h. Lumens are outlined in purple, green, and orange, respectively. b Quantification of cross-sectional luminal area of MDCK spheroids grown for 3 days in control, ddAVP, and ouabain conditions (p-values are from two-sided rank-sum test). c Quantification of mean cross-sectional cell thickness of MDCK spheroids grown for 3 days in control, ddAVP, and ouabain conditions (p-values are from two-sided rank-sum test). d Schematic describing solidity, a metric that reflects surface irregularity, and normalized lumen radius metric. e Solidity of lumens from MDCK spheroids grown for 3 days and 7 days, treated as indicated with vehicle (purple), ddAVP (green), or ouabain (orange) for 4 h as a function normalized lumen radius (ratio of estimated lumen radius determined by lumen volume and mean cell width) (p-values from two-sided 2D Kolmogorov–Smirnov test). f Representative images of fixed wildtype (WT, left) and Claudin-quintuple KO (Cldn-qKO, right) MDCK spheroids with lumens immunostained for the apical surface protein podocalyxin (PDX, orange) and DNA (blue)²⁶. g Lumen solidity plotted as a function of normalized lumen radius for wildtype lumens (WT, purple), Cldn-qKO lumens (green) (p-value from two-sided 2D Kolmogorov–Smirnov test). Scale bars are 10 μm. Box plots in b and c show median, quartiles of dataset, and whiskers extending to maximum and minimum of distributions, excluding outliers (indicated with diamonds). For plots b and c, n = 15, 21, 20 spheroids for control, ddAVP, and ouabain conditions, respectively. For plot e, n = 48, 33, 42 spheroids for control, ddAVP, and ouabain conditions, respectively. For plot g, n = 51, 25 spheroids for WT and Cldn-qKO conditions, respectively. Source data are provided in the Source Data file.