Figure 4. AA astrocytes exhibit increased migration compared to CA astrocytes.

A. Confirmation of three differentially expressed motility genes by qRT-PCR in human normal ONH astrocytes: autocrine motility factor receptor (AMFR), myosin light chain kinase (MYLK) and myosin phosphatase target subunit 2 (MYPT2). Genes were normalized to 18S. Graphical representation of the relative mRNA levels in AA and CA astrocytes (n = 8, respectively, * indicates p<0.05 in two-tailed t-test). B. Representative Western blots of astrocyte cell lysates with MYLK antibody. β-actin was used as a loading control. Note that AA1-4 donors express more MYLK 130 kDa than CA1-4 donors. No difference was detected at the levels of 210 kDa isoforms. C. Densitometry analysis of MYLK western blots. β-actin was used as loading control. Astrocytes derived from 7 AA and 10 CA were used in this experiment. The level of the 130 kDa isoform was significantly higher in AA astrocytes, compared to CA astrocytes. D. Cell migration assay shows that AA astrocytes migrate significantly faster than CA astrocytes. The assay was performed as described in the Materials and Methods. Values represent mean optical density (OD)±standard deviation of triplicate experiments using primary astrocyte cultures of six AA donors and five CA donors. * indicate p value<0.05. E. Inhibition of MYLK by ML-7 (10 µM), leads to a decrease in migration of African American ONH astrocytes (n = 3). F, G, H, I. Phalloidin staining of the actin cytoskeleton in normal AA (F) and CA (G) astrocytes. Inhibition of MYLK by ML-7, leads to a disruption of cytoskeleton in AA (H) and CA (I) ONH astrocytes. Magnification bar: 25 µm.