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. 2014 Feb 6;5(2):e1049. doi: 10.1038/cddis.2014.3

Figure 3.

Figure 3

Involvement of PKC phosphorylation and translocation. (a) The cells were incubated in the presence of Gal-1 (10 ng/ml) for varying periods of time (0–120 min) and then harvested. Total protein was extracted and blotted with phospho-PKC and total-PKC antibody. (b) The cells were incubated with 2 μM Fluo-3/AM in serum-free medium for 40 min and treated with Gal-1 (10 ng/ml) and were then treated with A23187 (10 μM, Ca2+ ionophore). Gal-1 and A23187-induced Ca2+ influx was measured. Changes in [Ca2+]i were monitored by confocal microscopy, and data are expressed as relative fluorescence intensity (F/F0%, arbitrary unit). (c) The cells were stimulated with Gal-1 (10 ng/ml) for 90 min, and extracted cytosolic and membrane proteins were then detected with PKC translocation of PKC isoforms. Western blot analysis showed that Gal-1 induced the membrane translocation of PKCα, δ, and ζ. (d) The cells were pretreated with β-lactose (10 mM) for 30 min before 10 ng/ml Gal-1 treatment for 90 min. Total protein was extracted and blotted with phospho- and total PKC antibody. (a, c, and d) Each example shown is representative of four independent experiments. The lower or right part of (a, c, and d) depicting the bars denote the mean±S.E. of four independent experiments for each condition determined by densitometry relative to β-actin or total-PKC. *P<0.05 versus control, **P<0.05 versus Gal-1 alone. (e) Oris cell motility assay. Cells were pretreated with 10 μM bisindolylmaleimide I and 10 μM staurosporine (PKC inhibitors) for 30 min before 10 ng/ml Gal-1 treatment for 24 h and stained with calcein AM (5 μM). Fluorescence in the analytical zone was quantified with a plate reader. Data represent means±S.E. of five independent experiments with triplicate dishes. *P<0.05 versus control, **P<0.05 versus Gal-1 alone. (f) In vitro UCB-MSC wound healing motility assay. Ten fields per plate were examined. Scale bars represent 100 μm (magnification × 100). Cytosol, cytosolic; Mem, membrane; PKC, protein kinase C; ROD, relative optical density