Extended Data Fig. 3: Bilirubin induced fusion requires close proximity and cytoskeletal signaling.
From: Multi-zonal liver organoids from human pluripotent stem cells
a) Brightfield images of bilirubin induced fusion in high density HLOs compared to low density and no bilirubin treatment. Scale bar indicates 200 µm. b) Comparison of mean segment length in high density HLOs compared to low density and no bilirubin treatment. (n = 9 independent experiments). c) Brightfield and live staining images (NucBlue: Blue, Cytoskeleton: White) show progression of organoid fusion after continued treatment with bilirubin (1 mg/L). Scale bar indicates 200 µm. (n = 3 independent experiments). d) Comparison of mean segment length of the HLOs from D1 to D7. (n = 9 independent experiments). e) NOTCH activity assay in bilirubin treated HLOs compared to control. (n = 9 independent experiments). f) Percentage of fused organoids after bilirubin treatment in DAPT (Notch inhibitor) and NSC (Ezrin Inhibitor, NSC668394) treated HLOs compared to control. (n = 9 independent experiments). g) Brightfield images of bilirubin induced fused HLOs compared to DAPT or NSC668394 treatment and control HLOs. Scale bar indicates 200 µm. (n = 3 independent experiments). h) CLF assay for self-assembled organoids compared to control. Scale indicates 200 µm. (n = 3 independent experiments). i) Percentage of fused organoids for each type of organoid. (n = 9 independent experiments). In e data is represented as boxplot where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper and lower whisker extends from the hinge to the largest and smallest value respectively no further than 1.5 × IQR from the hinge (where IQR is the inter-quartile range). b,d, Kruskal-Wallis with multiple comparisons and Dunn-Holland-Wolfe correction. e, unpaired two-tailed Student’s t-test. i, one-way ANOVA with multiple comparisons and Tukey’s correction.
