Fig. 2: UXT overexpression protects neuronal cells from proteasome inhibition-induced cell death.

a Immunocytochemistry of dorsal root ganglion (DRG) neurons. DRG neurons were treated with 2.5 µM MG132 for 12 h and compared to untreated controls. Endogenous p62 and ubiquitin-positive protein aggregates (upper panels) or p62 and UXT (lower panels) were stained under both conditions with their specific primary antibodies, and subsequently with anti-rabbit IgG-TRITC and anti-mouse IgG-Alexa-350 as secondary antibodies. Scale bar, 10 µm. b On day 0, DRG neurons (white arrows) were isolated from day 13.5 mouse embryos and cultured for 48 h. On day 2, mCherry or mCherry-UXT was introduced into DRG neurons by lentiviral transduction and cells were cultured for 2 days. On day 4, the DRG neurons were treated with 2.5 µM MG132 and observed under a live imaging microscope with temperature and humidity control. DIC (differential interference contrast) images were collected for 28 h after MG132 treatment at 20 min intervals (Supplementary Movie 1). To identify the transduced cells, fluorescence images were also taken at the end. c DIC images collected at 0, 8, 18, and 24 h and mCherry fluorescence images of DRG neurons are shown. Transduced cells are indicated with red asterisks. Membrane blebs are indicated with arrowheads. Scale bar, 20 µm. d Survival of DRG neurons transduced with mCherry without MG132 treatment (blue), mCherry with MG132 treatment (black), and mCherry-UXT with MG132 treatment (red) was analyzed and is shown with the number of DRG neurons analyzed. This analysis was repeated twice with the essentially same results.