Effect of proteasome inhibition by PSI on both cell survival and formation of PSI-induced inclusions in PC12 cells, and assessment of a three-process fractionation procedure on isolation of PSI-induced inclusions from the cells. A, trypan blue staining of cells: a, cell survival (open arrows) alone in cells exposed to DMSO vehicle for 48 h; b, cell survival and addition of cell death (closed arrows) in cells exposed to 10 μM PSI for 48 h; c, percentages of the number of living cells. B, H&E histological staining of inclusions: a, cells in the vehicle; b, nucleus-binding (open arrows) and nucleus-free (closed arrows) inclusions formed in cells in 10 μM PSI for 48 h; c, the two morphological types of inclusions in the fraction of initial pellets; d, percentages of nucleus-free to total inclusions in the cells in PSI (Column 1) and in the fraction of initial pellets (Column 2); e-h, the one (or two) morphological type (s) of inclusions in the first four temporary fractions of resulting pellets; i, the one morphological type of inclusions in the final fraction of resulting pellets; j, percentages of nucleus-free to total inclusions in the five fractions of resulting pellets (Column 3-7). C, alpha-SYN immunostaining for and H&E histological staining of inclusions: a and a', cells in PSI; b and b', an isolated fraction of inclusions. D, immunoblotting for histone H1 in inclusions: a, bands of histone H1 in gel image; b, quantitative level of histone H1 in densitometry. Scale bar, 10 μm.