Gelred

Sixty worms of the strain UA196 [sid-1(pk3321); P_dat-1::α-syn, P_dat-1::GFP; P_dat-1::sid-1, P_myo-2:: mCherry] were grown to day 7 at 20°C. They were harvested from drug treatments or solvent control plates (30 μM MFX/0.1% DMSO; 30 μM CsA/0.1% DMSO; 30 μM SUL/0.1% DMSO; 10 μM CC/ddH₂O). Worms were transferred to fresh drug/RNAi plates as needed to avoid starvation. Total RNAs were extracted from control and drug-treated worms as described previously [58 (link)]. Total RNA was quantitated using a Nanodrop and 1 μg of total RNA of each sample was used to synthesize first-strand cDNA using MMLV-RnaseH(-) transcriptase (Promega). Using the cDNA as templates, PCR reactions were conducted with GoTaq polymerase (Promega) at 59°C annealing temperature. Primer sequences are as follows:
PCR products were loaded onto a Gel Red (Sigma) stained 0.8% agarose gel. An image was captured by FujiFilm LAS 4000. Band intensities were compared by digital imaging using MetaMorph software. Fluorescent band intensities were normalized using the following equation: α-syn/ama-1.

HEK 293T cells were cultured in large quantities, and the lysates underwent subcellular fractionation to obtain a nuclear pellet, which was then dialysed. The pBSK (+) duplex plasmid DNA was linearised with EcoRI and then co-incubated with nuclear extracts, in which genomic DNAs were fragmented into small pieces (<500 bp) by sonication, in reaction buffer at 37 °C for 1 h according to previously described methods^{22 (link),23 (link)} and then subjected to 0.7% agarose gel electrophoresis after deproteinisation (10 μg/μl proteinase K, 3% SDS, 50 mM EDTA and 100 mM Tris-HCL, pH 7.5, 90 min at 57 °C). The gel was then incubated with the highly sensitive nucleic acid dye GelRed (Sigma-Aldrich) at 1:10,000 dilution in water at room temperature for 15 min and then de-stained in water for ~1 h. The Quantification of the DNA bands was performed using a phosphorimager (Analytik Jena AG).