Cells were incubated with 100 μl of radioimmunoprecipitation assay (RIPA) buffer containing protease and dephosphatase inhibitors (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at 4°C and then centrifuged at 16,000 × g for 20 min. Total protein was then subjected to immunoblotting using antibodies against Cleaved caspase 3 (1:500), H3K9me2 (1:200; Merk), Histone 3 (1:1,000; Merk), Nestin (1:500), NF-M (1:500), MAP-2 (1:500), GAPDH (1:1,000; Santa Cruz Biotechnology), or β-actin (1:5,000; Sigma-Aldrich), followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Details of primary and secondary antibodies are summarized in Supplementary Tables 3 and 4.