Skip to main content
. Author manuscript; available in PMC: 2015 Dec 17.
Published in final edited form as: Neuron. 2014 Dec 17;84(6):1213–1225. doi: 10.1016/j.neuron.2014.12.010

Figure 4. PR50 nuclear aggregates co-localize with nucleolin and mediate cellular stress responses.

Figure 4

(A) Cortical neurons transfected with a combination of R42 and PR50 constructs (0.4 μg R42 + 0.4 μg PR50 + 0.2 μg Td-tomato; total 1 μg/well cDNA) displayed significant increase in cumulative risk of death compared to the other three possible combinations, suggesting R42 and PR50 increased neural toxicity synergistically. The cDNA concentration for each toxic species is 50% of the concentration used when R42 or PR50 were transfected alone in other experiments. At least 80 neurons were imaged/group; n=3 experiments. **P<0.01; ***P<0.001. (B) The combination of GA50 and R42 did not have synergistic effects. P=0.08. (C) Immunofluorescence analysis of cortical neurons transfected with PR50 revealed that PR50 nuclear aggregates co-localized with nucleolin in the nucleolus (bottom panels), and PR50 aggregates induced (D) dispersal of nucleolin with increase in nucleolus size. DAPI: Blue; GFP control or PR50: green; Nucleolin: red. Calibration bar is 10 μm. (E) Neurons transfected with PR50 showed decreased number and increased size of P-bodies. Representative images of GFP control or PR50 transfected cortical neurons. DAPI: blue; Dcp1: white; GFP control or PR50: green. DCP1 is the mRNA-decapping enzyme 1A, constituent of P-bodies. Calibration bar is 10 μm. (F) Quantification of number and size of P-bodies in control and PR50 transfected cortical neurons displaying diffused and aggregated PR50. Diffused PR-staining was previously associated to neuronal survival. At least 20 neurons/group were counted per experiment; n=3. (***P<0.001; two-tailed t-test). Calibration bar is 10 μm. (G) Stress granules formed only in motor neurons in which PR50 aggregated (arrow). DAPI: blue; TIAR: red; GFP control or PR50: green; MAP-2: white. TIAR = TIA1 (cytotoxic granule-associated RNA binding protein-like 1). Calibration bar is 10 μm.