Figure 2

(A) Human CECs were seeded for 24 hours before exposure to various concentrations of Y-27632 (n = 7) including 3 µM, 10 µM, 30 µM*, 100 µM**, 300 µM** and 1000 µM** for another 24 hours and analyzed using the xCELLigence system. (B) Normalized impedance readings of CECs treated for 24 hours, in various concentrations of Y-27632 (solid bars) and their respective recovery at Day 5 after the withdrawal of Y-27632 at Day 2 (striped bars). Cell index was normalized to 1 (dotted line) to untreated controls at 24 hours and at Day 5. (C) Representative time-lapse micrographs of CECs at start state, before the addition of Y-27632; 1 hour and 24 hours after the addition of Y-27632; and 24 hours after Y-27632 withdrawal. Either 10 µM (top panel) or 1 mM (bottom panel) of Y-27632 was used. (D) Impedance readings of CECs adherence at 4 hours, with or without the presence of 10µM of Y-27632 (n = 4) were measured using xCELLigence. Representative phase-contrast micrographs showing the morphology of CECs at 4 hours following cellular attachment (left panel) and at confluence (right panel), with insert showing a higher magnification of the confluent culture of (E) normal control P0 CECs and (F) their donor-matched counterparts which were exposed to 10 µM of Y-27632 throughout culture. (G) Overall cellular yield of isolated donor-matched P0 CECs growth to confluence with or without exposure to 10 µM of Y-27632. (H) Morphometric analysis (n = 3) – cell size*, cell circularity and coefficient of variance – of confluent donor-matched P0 human CECs grown with or without any exposure to 10 µM Y-27632 (*p < 0.05 and **p < 0.01) – For (D) and (H), ‘C’ denote control; ‘Y’ denote Y-27632 treated. Scale Bars: 100µm.