DAPI was used to stain the nucleus. 20 m. (D) Confocal microscopy imaging of cortical neurons co-transfected at DIV10 with the indicated DRP and synapsin-driven Td-tomato construct to aid visualization of neurons. Cortical neurons were imaged 48 hours post-transfection. Expression levels of Td-tomato reporter GSK690693 protein were noticed to be consistently lower in PR-expressing neurons. Exposure settings were uniform throughout confocal analysis. Arrows point at aggregates within the soma and neurites (GA50) and nucleus (GR50 and PR50). Calibration bar is 20 m. (E) DRP constructs (50 repeats long) in pcDNA3.1 vectors were transfected in primary rat motor neurons at DIV5 (0.5 g DNA/well). Neurons were imaged by confocal microscopy 24 hours post-transfection. Calibration bar is 20 m. Arrows point at aggregates within neurites (GA50) and nucleus (GR50 and PR50). Calibration bar is 20 m. Figure S3. Time-lapse imaging of individual neurons in culture. (A) Schematic of live-cell longitudinal tracking experiments. Primary cortical neurons are transfected at DIV 10 and transfected neurons (Td-tomato+) in the same optical field are imaged at a 24h interval for up to 9 days post-transfection. To monitor individual neurons, a synapsin promoter driven Td-Tomato construct was co-transfected as a sensitive reporter of survival. (B) Representative images of cortical neurons co-transfected with Td-tomato reporter construct driven by synapsin promoter and GFP-control construct. Calibration bar is 100 m. (C) Cortical neurons were imaged at transfection day 0 in bright field, which corresponds to DIV10. Image shows full maturation of the cortical neurons in vitro. The same neurons in the optical field were then imaged for 8 consecutive days post-transfection at 24-hour intervals using Td-tomato as fluorescent reporter. Arrows point at 4 neurons that were successfully transfected in this field. Time-lapse images show the increased expression of the Td-tomato reporter construct in those neurons that allows visualization of the in vitro neurites network. Calibration bar is 100 m. Figure S4. Survival analysis of different DRPs transfected in motor and hippocampal neurons. (A) Representative live-cell images IL6 antibody of motor neurons co-transfected with Td-Tomato (0.1 g/well; red fluorescence signal shown in top panels) and PR50 cDNA plasmids (0.4 g/well; green fluorescence in bottom panels). Motor neuron with aggregates died, while motor neuron with diffused PR50 expression (orange arrow and inlet) did not undergo neurodegeneration. Calibration bar is 20 m. (B) Kaplan-Meier survival analysis suggested that both PR50 and GR50 were toxic to primary motor neurons compared to control (***P 0.001). Although a trend was observed for GA50 GSK690693 expressing motor neurons difference with control did not reach significance. At least 40 neurons were followed/group; n=3 independent experiments. (C) Kaplan-Meier survival analysis of hippocampal neurons transfected with different DRP constructs showed that PR50 was toxic to hippocampal neurons. At least 80 neurons were followed/group; n=3 independent experiments. (D) However, hippocampal neurons were less vulnerable to PR50 compared to cortical neurons. *P 0.05, ***P 0.001. (E) Expression levels of GFP were quantified 72 hours post-transfection by confocal microscopy measuring immunofluorescence intensity in each neurons. At least 20 cortical and hippocampal neurons were imaged and quantified (Image J). Camera acquisition parameters were set the same between the two groups (unpaired t-test; P=0.0824). (F) Expression of untagged PR50 causes cortical neuron death, which is not significantly different from that caused by GFP-PR50 as shown by Kaplan-Meier survival analysis. At least 40 neurons/group; n = 4C6 GSK690693 experiments; ***P 0.001. (G) Immunoflurescence analysis using -PR antibody shows that untagged PR50 forms nuclear aggregates. Td-Tomato signal indicates a neuron transfected with untagged PR50..