Fig. 3. CD13 is recruited to the apical membrane-initiation site before Par6 in 3D Caco-2 cysts.

a Confocal images of 2-cell Caco-2 spheroids immunostained for CD13-V5 (green) and Par6 (magenta) showing the localization of CD13 and Par6 at adhesion, AMIS, and PAP stages. Middle - profiles depicting fluorescent intensity (8-bit) of CD13 and Par6 along the cell-cell edge from blue to yellow arrows. Right - Scatter plots showing the relationships between Par6 and CD13 pixel intensities. b Confocal images from Caco-2 cells immunostained for endogenous CD13 during polarization and early lumen formation stages (1-cell, adhesion, AMIS, PAP, lumen). c, d Caco-2 cells were lentivirus-infected to express CD13-mCherry and EGFP-Par6 vectors in 2D culture, then transferred to 3D culture to initiate cell polarization. Time-lapse confocal images were captured every 25 min. Between adhesion and AMIS stages, CD13 disperses into puncta that coalesce into an AMIS that subsequently opens to form a lumen (c). During coalescence, CD13-puncta accumulation precedes incorporation of Par6 at the AMIS (d). Images are representative from three independent experiments. Bars: a–d, 10 μm.