A. Western blots of Rap1a/b and Afadin in Rap1a/b double knockdown (Rap1 DKD) and double knockout (Rap1 DKO) cells.
B. Immunofluorescence of Podxl1, cadherin-6, and ZO1 in 2-day and 5-day spheroids with Rap1 DKD and DKO. The Rap1 DKD/DKO spheroids have multiple lumens. Scale bars: 10 μm.
C. Quantification of percentage of spheroids with a single lumen at 5 days. Rap1a/b DKD and DKO spheroids have a lower percentage with a single lumen. Results from two double DKO clonal lines are shown.
D. Quantification of the percentage of two cell spheroids at the indicated lumen stages at 20h. See Fig 1A and text for details. Rap1a/b DKO spheroids have fewer in the AMIS and lumen stages, indicating delay in lumen formation.
Results are representative of at least 2 independent experiments.