Integrin Linked Kinase (ILK) regulates astrocytic differentiation by NSCs.

<p>A. NSCs cultured in either control (scrambled) PA (VVIAK PA) or IKVAV PA for one day, and immunostained with activated β1integrin antibody HUTS-4 (green) or ILK (red). <i>Scale bar in HUTS-4 staining image = 10 µm, and in the ILK staining image = 20 µm.</i> B. Western blot analysis of NSCs cultured in either control PA (VVIAK PA) or IKVAV PA for one day. Note that levels of ILK are markedly elevated in the IKVAV-PA group without any change in FAK. C. β1-integrin KO and control NSCs were transfected with either a control construct or a construct expressing a constitutively active form of ILK (ILK-CA). At 7DIV the cells were immunostained for GFAP and numbers of GFAP⁺ cells were counted. (*p≤0.05) D. β1-integrin KO and control NSCs were transfected with a control construct, a construct expressing constitutively active ILK (ILK-CA), or a construct expressing kinase-dead ILK (ILK-KD) and cultured for 7DIV. RNA was extracted for qPCR measurement of GFAP mRNA graphed as a ratio compared to the control (**p≤0.004). Note that β1-integrin KO increased levels of GFAP mRNA, but this increase was blocked by expression of ILK-CA. E. Western blot analysis of GFAP expression in control NSCs and in β1-integrin KO NSCs transfected with either with a control construct or ILK-CA. Note that transfection with ILK-CA prevented the increase in GFAP expression in β1-integrin KO NSCs. F. NSCs were treated with different doses of the ILK inhibitor, Cpd22. After 6DIV RNA was extracted for qPCR quantification of GFAP. Note that GFAP expression increased in a dose dependent manner after treatment with Cpd22. H. NSCs were treated with different doses of the ILK inhibitor, Cpd22 for 3h, and protein was extracted for western blot analysis. Note that treatment with the drug reduced levels of phospho-Akt without any change in levels of AKT.