44p software. The neurite length http://www.selleckchem.com/products/MG132.html and diffe rentiation rate were evaluated according to the following definition the length was the straight line distance from the Inhibitors,Modulators,Libraries tip of the neurite to the junction between the cell body and neurite base. In the case of branched neurites, the length of the longest branch was measured. For each cover glass, 20 and 40 images were acquired randomly by scanning the wells, measuring in each image N, as total number of cells. n, as number of cells with the neurite longer than 20 um. l, as neurite length in um. R, as diffe rentiation rate determined by the equation R 100 n N. Cell spreading assay For each cover glass, 10 and 20X images were acquired randomly by scanning the wells and the cell density for cm2 was measured.

Neurite length is presented Inhibitors,Modulators,Libraries as arithmetic mean normalized for not differentiated cell number. Each substrate type was tested Inhibitors,Modulators,Libraries 3 times with at least 100 cells considered. All data are expressed as Inhibitors,Modulators,Libraries sample arithmetic mean S. E. M. Signifi cance of differences was determined using one way ANOVA and Tukey post hoc test. Immunofluorescence staining Immunofluorescence studies were performed after 48 h from PC12 cells culture on flat TiO2 substrate, ns TiO2 substrates and PLL glass. Samples were fixed and immunostained for F actin using an AlexaFluor555 Phal loidin probe. Briefly, at room temperature cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. after washing, cells were permeabilized with permeabilization buffer containing 0. 2% BSA and 0. 1% Triton X 100 for 1 5 min, blocked with 2% BSA for 1 h, stained for actin for 40 min at room temperature.

Samples were Inhibitors,Modulators,Libraries rinsed twice with PBS and nuclear labeling was performed by 4. 6 diamidino 2 phenylindole. Samples were rinsed twice with PBS, mounted with 90% glycerol and sealed. Fluorescent images were worldwide distributors obtained with a Leica Confocal Microscopy TCS SP2. Lysate preparation and Western blot analysis For preparation of whole cell extracts, cells from cul tures exposed to NGF from zero to 2 days were washed with PBS and extracted for 10 min at room temperature with sodium dodecyl sulfate polyacrylamide gel electropho resis sample buffer, then the fraction was col lected. To separate cytosolic and cytoskeletal associated proteins cells were washed with PBS and extracted for 10 min at room temperature with PEM buffer containing 0. 1% v v Triton X 100, then the frac tion was collected. The obtained Triton X 100 soluble frac tions were diluted 3 1 with 4X SDS PAGE sample buffer. The insoluble material remaining attached to the dish was scraped into SDS PAGE sample buffer. Equal proportions of each fraction, representing proteins from the same number of cells, were separated by SDS PAGE.