Figure 3: p68 and CaM interaction is essential for cell migration and for CaM localization.

(a, upper) Levels of p68 in the p68 knockdown SW480 cells were examined by immunoblot in whole-cell lysates (WCLs). (a, bottom) Levels of exogenously expressed HA-p68s, WT and indicated mutants, were analysed by immunoblots of WCLs using anti-HA antibody. Immunoblots of GAPDH and β-actin are loading controls. (b) Migration of SW480 cells in which endogenous p68 was knocked down by RNAi and HA-p68s, WT and mutants (indicated), were exogenously expressed, was analysed by Boyden chamber assays. The number of cells migrating to the lower chamber was measured relative to the number of migrating cells in which the empty vector was transfected. (c, left panel) Migration of SW480 cells in which endogenous p68 was knocked down by RNAi and HA-p68s, WT and mutants (indicated) were exogenously expressed, revealed by phase contrast microscopy 0 and 24 h after scratch-wounds were introduced into the cell culture plates. (a, right panel) Quantification of the cells in a fixed area that migrated to the scratch area. (d) Representative images of SW480 cells in which endogenous p68 was knocked down by RNAi and HA-p68s, WT and mutants were exogenously expressed. HA-p68s was immunostained using anti-HA antibody (green). Endogenous p68 was stained with the antibody p68-RGG (green). 4′,6-Diamidino-2-phenylindole (blue). (e) Representative images of immunofluorescence staining of p68 (red) and CaM (green) in SW480 cells. The cells were treated by scratch-wounding. Upper panel shows cells that located to the scratch edges, bottom panel shows the middle of scratched areas. (f) Representative images of immunofluorescence staining of p68 (green) or exogenously expressed HA-p68s (green), WT or mutants (indicated), and CaM (red) in SW480 cells. The endogenous p68 was knocked down by RNAi. The cells were treated by a scratch-wound (scratch) or no scratch-wound (no scratch) in the culture plate. Error bars in b and c are standard deviations of three independent measurements. Vec is the cells transfected with the empty vector. NT represents the cells treated with non-target RNAi. Scale bars, 100 μm (for c) and 20 μm (for d,e and f).