Fig. 6: ANXA1 is required for correct mitotic spindle orientation during 3D morphogenesis.

a Confocal images of representative mouse mammary gland cryosections (50 µm-thick) stained for ANXA1 (green), K14 (grey) and E-cadherin (magenta). b Confocal images of representative mMEC acini stained for F-actin (green) and α-tubulin (grey) or Par6 (green) and counterstained with DAPI (DNA, magenta). c Spindle angle frequencies and mean angles (mα) (3 independent experiments, 48 h: n = 42 acini; 72 h: n = 40 acini; 96 h: n = 39 acini). d Confocal images of representative acini stained for ANXA1 (green) and counterstained with DAPI (DNA, magenta). e Western blotting of extracts from shRNA-transduced acini. f Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for F-actin (green) and α-tubulin (grey) and counterstained with DAPI (DNA, magenta). g Spindle angle frequencies and mean angles (mα) (3 independent experiments, sh-Control: n = 30 acini; sh-ANXA1#1: n = 30 acini; sh-ANXA1#2: n = 30 acini). Kolmogorov-Smirnov test, **P < 0.01. h Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for LGN (green) and counterstained with DAPI (DNA, magenta). i Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for F-actin (green) and E-cadherin (grey) and counterstained with DAPI (DNA, magenta). j Percentage of acini with normal lumen (left) (4 independent experiments, sh-Control: n = 163 acini; sh-ANXA1#1: n = 142 acini; sh-ANXA1#2: n = 168 acini), acinus area (middle) and acinus roundness (right) (3 independent experiments, sh-Control: n = 30 acini; sh-ANXA1#1: n = 30 acini; sh-ANXA1#2: n = 30 acini). One-way ANOVA with Tukey’s test, left: ***P = 0.0008 and ***P = 0.0005; middle: P = 0.201 and *P = 0.067; right: **P = 0.007 0.01 and **P = 0.002. k Number of lumen per acinus (left), lumen area (middle) and lumen circularity (right) (3 independent experiments, sh-Control: n = 36; sh-ANXA1#1: n = 32; sh-ANXA1#2: n = 31). One-way ANOVA with Tukey’s test, left: ****P = 0.00009 and ****P = 0.00002; middle: ****P = 0.0001 and ****P = 0.00007; right: ****P = 0.0001 and ****P = 0.00006. l Left: illustration showing quantification of cortical fluorescence intensity of F-actin (green) and E-cadherin (grey) in acini. Right: average cortical fluorescence intensity profiles of E-cadherin and F-actin (3 independent experiments, sh-Control: n = 30; sh-ANXA1#1: n = 30; sh-ANXA1#2: n = 30). All time-points of 3D culture are indicated in hours (h). All data are presented as mean ± s.e.m. n.s. (not significant). arb. units (arbitrary units). All scale bars, 10 µm. Source data are provided as a Source Data file.