Upright fluorescence confocal microscope

To analyze mitochondrial distribution, treated-PCCN cells were incubated with pre-warmed (37°C) staining solution containing the Mitotracker Green FM probe (M7514) (Invitrogen) (final concentration 100 nM) for 45 min. After staining is complete, the staining solution was replaced with prewarmed fresh media and observed using an upright fluorescence confocal microscope (Olympus, Tokyo, Japan). The number of cells with non-tubular/fragmented mitochondria was determined, with at least 500 transfected cells examined. All quantifications were based on at least three independent experiments.

The pCMV-HA-Rest vector of full-length REST (Cat No. PPL50007-2a) was obtained from the Public Protein/Plasmid Library (Nanjing, China, GeneShare Technology, co, Ltd). The pGPH1/GFP/Neo-REST-Rat short-hairpin RNA (shRNA) against Rat REST were obtained from GenePharma (Suzhou, China). Mature antisense sequences of effective shRNAs were 5′-GCTGTGGCTACAATACCAACC-3′ (946-966) (shREST-1) and 5′-GTGCAATTATGTGGCCTCTAA-3′ (1295-1315) (shREST-2). For transfection (Song et al., 2014 (link), 2016 (link); Zhu et al., 2015 (link)), cultured primary neurons were washed with Opti-MEM (Invitrogen) in 12-well plates and then transfected with the appropriate plasmids using the Lipofectamine 2000 reagent (Invitrogen) in serum-free Opti-MEM according to the manufacturer’s instructions. The amounts of plasmids and reagents were 2 μg and 3 μl per 12-well, respectively. The culture medium was replaced by the primary cell culture medium 5 h after transfection. Forty-eight hours after transfection, the cells were observed under an upright fluorescence confocal microscope (Olympus, Tokyo, Japan) or subjected to immunoblot analyses.