Generation of More Than Two Centrosomes in vHMECs following S Phase Arrest Is Due to Centriole Pair Splitting

(A and B) Analysis of the centriole number in of the supernumerary centrosomes of early-passage vHMECs (RM15 [19 PD]) that express EGFP-CETN2 (centriole marker, green) untreated (−HU) or exposed to HU (+HU). Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein (centrosome marker, red). Analysis included at least 100 cells (excluding binucleated cells). *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments.

(B) Examples of HU-exposed vHMECs with one centrosome (containing a pair of centrioles), two centrosomes (each containing one centriole), three centrosomes (one of the centrosomes contains a pair of centrioles and two of the centrosomes have only one centriole), and four centrosomes (each containing one centriole).

(C) Examples of HU-exposed and released vHMECs that express EGFP-CETN2 (green) and have been stained with a γ-tubulin antibody that recognizes microtubule spindles (red) that have two centrosomes, each containing two centriole (top), and supernumerary centrosomes, each containing one centriole (bottom). Supernumerary centrosomes with one centriole (arrowhead) can nucleate microtubules to form a multipolar spindle apparatus.