HEK293 cells (from Leibniz-Institut DSMZ, Braunschweig, Germany) were transfected with polyethyleneimine and 2 μg of plasmid DNA in serum-free medium. After 5 hours, complete medium was added. Cells were transfected with the following constructs: GFP (as control), SRF-VP16, SRF-VP16 ΔMADS (44 (link)), constitutively active MRTF-A (52 (link)), Poly-GA (75 (link)), SOD1G93A (47 (link)), SOD1A4V (45 (link)), or P62-GFP-mCherry (46 (link)). For the Poly-GA–lysotracker experiment, after overnight incubation, 1 μM Lysotracker Deep Red (Thermo Fisher Scientific) was added and incubated for 2 hours at 37°C. For qPCR experiments with HEK293 cells, RNA was extracted with TRIzol (Qiagen). For reverse transcription, 1 μg RNA was mixed with random primers dN6 (Biomers), incubated for 10 minutes at 70°C, and placed on ice. Then, the master mix (room temperature 5× buffer, Promega Biosciences), dNTPs (Genaxxon), Ribolock RNase Inhibitor (Thermo Fisher Scientific), and M-MLV RT RNase (Promega Biosciences) were added. After 10 minutes at room temperature, the mixture was incubated at 42°C for 45 minutes, at 99°C for 3 minutes, and, thereafter, on ice.
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