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. 2013 May 13;288(26):18853–18862. doi: 10.1074/jbc.M112.443671

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 8. — Role of secreted cystatin C in stimulating NSPC proliferation. The number of viable cells was calculated from the fluorescence intensity in an alamarBlue assay. A, effect of cystatin C on NSPC proliferation. Dissociated neurosphere-derived cells were plated on poly-l-lysine-coated plates and then incubated for 5 days in proliferation medium containing various concentrations of cystatin C. Values are means ± S.E., (n = 4) and shown as the percentage of the control value (no added cystatin C). * = significantly different from the control group (no cystatin C) (p < 0.05, ANOVA with post hoc Tukey's test). B, Western blot analysis of cystatin C (CysC) immunoreactivity in cell culture medium. Panel i, comparison of the level of cystatin C in 48 μl of C57Bl/6xSJL cell medium when compared with 100 ng of recombinant cystatin C. Panel ii, Western blot stained for cystatin C showing efficiency of immunodepletion of cystatin C from the C57Bl/6xSJL and Tg2576 conditioned medium. Ig = medium after immunodepletion with control immunoglobulin; CC = medium after immunodepletion with cystatin C antibody. Panel iii, Western blot stained for cystatin C showing efficiency of immunoprecipitation of cystatin C from the C57Bl/6 and APP-KO cell medium. C and D, immunoprecipitation of cystatin C removes the APP-induced growth factor that stimulates proliferation. C, C57Bl/6xSJL neurosphere-derived cells were incubated with unconditioned medium or conditioned medium from C57Bl/6xSJL cultures or Tg2576 cultures that was previously immunoabsorbed with a nonspecific Ig fraction (IP: Ig) or with an anti-cystatin C antibody (IP: CC). D, APP-KO neurosphere-derived cells were incubated with unconditioned medium or conditioned medium from C57Bl/6 cultures or APP-KO cultures that was previously immunoabsorbed with a nonspecific Ig fraction (IP: Ig) or with an anti-cystatin C antibody (IP: CC). Values in panels C and D are means ± S.E. (n = 4) and shown as the percentage of the mean of the values for unconditioned medium. * = significantly different (p < 0.05, ANOVA with post hoc Tukey's test).