Figure 4

Cyclin B staining and analysis of passage through G2 by IdU pulse-chase. FACS analysis of co-staining for CD44 and cyclin B1 of CaLH3 cell line (A): panels A1 and A2 show gates positioned to identify CD44^highand cyclin B1 positive cells respectively; panels A3 and A4 show the distribution of cyclin B1 positive cells in CD44^high and CD44^low cell fractions respectively (p < 0.01). The same assay for the Ca1 cell line indicated 49.0 ± 3.5% of CD44^high cells expressing cyclin B1 compared to 14.9 ± 0.2% of CD44^low cells (p < 0.01). FACS plots of H357 cell line triple stained for DNA content with DAPI and with antibodies against CD44 and IdU adducts showing cell cycle distribution of IdU labelled cells in CD44^high and CD44^low cell fractions with time after labelling (B, C, D). Typical cell cycle distribution of total cell population at selected time points (B). Cell cycle distribution of CD44^low cells exposed to IdU for 60 min prior to fixation (0 hours) showed cells distributed in S and early G2. By 5 hours after labelling, cells began to return to G1, and by 7 hours nearly 40% of cells have re-entered G1 (C). Distribution of IdU-labelled CD44^high cells after initial labelling (0 hours) showed cells distributed in S and G2 as for CD44^low cells. By 5 hours after labelling fewer cells have returned to G1 and by 7 hours the number was still lower (D). Data for all time points are summarised for H357 (E) and CaLH3 (F) cell lines.