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Fig. 7. Inhibition of ROCK1/2 or myosin II promotes both PAP formation and lumen initiation. (A) Normalized probabilities of PAP formation (x) and lumen initiation (y) in 24 hour 3D cultures treated as indicated. Values were normalized to medium (control, Y-27632) or DMSO (DMSO, ML-7, blebbistatin) values. Values (± s.e.m.) that are significantly different from control (medium or DMSO; P<0.05) are indicated with asterisks. The values were calculated from frequencies of aggregates with PAP or lumen (see Materials and Methods) (supplementary material Fig. S5A,B). (B) Schematic representation of lumen formation as a linear sequence of events. The probabilities (see Materials and Methods for calculation) of PAP formation (x) and lumen initiation (y) in cultures treated with ML-7, Y-27632, or blebbistatin relative to control values (medium or DMSO) are shown in A. (C) Western blot analysis of myosin light chain phosphorylation after ML-7 and Y-27632 treatment. Depicted are levels of pMLC and ppMLC in monolayer cultures treated for 24 hours as indicated; β-actin levels as input control (see also supplementary material Fig. S5C). (D) Frequencies of aggregates with lumen in 96-hour cultures. The cells were treated with ML-7, Y-27632, or blebbistatin during the final 24 hours of culture, or were left untreated (control). The average frequency values are shown (± s.e.m.; n=50, three experiments; see also Materials and Methods).
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