SUPPLEMENTAL FIGURE LEGENDS
Supplemental figure S1: Degradation of nucleoporins in vitro and in primary dissociated
neurons upon glutamate treatment requires calpain activity. A) CGNs 7DIV were bathed in
MgCl2 and glucose-free/ Gly-containing CSS5 buffer in the presence of 10M glutamate.
After 30 minutes of oxygen deprivation, the cells were incubated with glucose and 2mM
MgCl2 for 3h (lanes 2,3) or 4h (lanes 4,5). As a control, oxygen deprivation was followed by
re-oxygenation for 4h (lane 1). Lanes 3 and 5 were neurons treated with calpeptin. B) Murine
cortical neurons 8DIV were challenged with glutamate for different periods of time, in the
presence or absence of calpeptin (90 minutes). Immunoblot analysis was performed using an
antibody to Nup93 and Mab414 as control. C) Incubation of homogenates of 293 T cells
overexpressing C-terminally Myc-tagged Nup93 with recombinant calpain II in the presence
of calcium. Immunoblot analysis was performed using antibody to Myc-tag. D) HeLa cells
were transfected with RNAi scramble oligo or duplex against Capn4 and Nup62. Immunoblot
analysis was performed using Mab414 and antibodies to Capn4 and -tubulin. E) HANup62(1÷100)-YFP or HA-NES (as control) were overexpressed in 293T cells and whole
cellular homogenates were incubated with recombinant calpain I, in the presence or absence
of Ca2+. Western blot analysis was performed with antibodies to HA-tag and GFP.
Supplemental figure S2: Localization of fluorescent reporters during neuronal death. A)
Schematic diagram of NES-2 and NES-3. The calpain cleavage site in NES-3 is indicated (^).
B) Whole cell homogenates of NES-2 overexpressing 293T cells were incubated with
recombinant calpain II, in the presence or absence of CaCl2. Immunoblot analysis was
performed using antibodies to fodrin and GFP. C) CGNs transduced with lentiviral particles
encoding NES-2 were exposed to excitotoxic glutamate concentrations for 60 or 90 minutes,
in the presence or absence of 5M calpeptin. Blot was probed with anti-GFP and anti-actin
antibodies. D) CGNs overexpressing NES-3 were treated with glutamate for the time
indicated, in the presence or absence of calpeptin. Western blot analysis was performed using
an anti-GFP antibody. E) NES-3 was overexpressed in CGNs by lentiviral transduction.
Upon glutamate stimulation, cells were fixed and stained with anti-GFP antibody (green).
Hoechst-33342 was used to visualize the nucleus (blue). Arrows indicate neurons presenting
nuclear accumulation of NES-3. F) Time lapse confocal microscopy of CGNs overexpressing
NES-3 and loaded with Fura-Red. Calcium homeostasis and nuclear accumulation of
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fluorescent probes were recorded simultaneously after challenging cells with 150M
glutamate. G) Whole extracts of NLS-YFP overexpressing 293T cells were incubated with
recombinant calpains, in presence or absence of calcium. Immunoblot assay was performed
with antibodies to fodrin and GFP. H) Immunoblot analysis using anti-GFP antibody of NGY
overexpressing CGNs, treated or untreated with glutamate. J) NGY was overexpressed in
293T cells and whole cellular homogenate was incubated with recombinant calpain II, in the
presence or absence of calcium. Western blot analysis was performed using antibodies
against GFP and fodrin.
Supplemental figure S3: Downregulation of selected nucleoporins affects the localization of
4xCherry. A) Immunoblot analysis of HeLa cells transfected with double-stranded RNA
oligonucleotides. Protein levels of Nup153 and Nup62 were analyzed using anti-Nup153 and
Mab414 antibodies, respectively. B) Confocal images of HeLa cells overexpressing 4xCherry
and transfected with RNAi duplex against nucleoporins.
Supplemental figure S4: Localization of fluorescent proteins in wtN2 and deg-3(u662)
animals. A) Confocal analysis of wt N2 animals overexpressing NLS-Cherry (red) and the
co-injection marker GFP (green) driven by Pmyo-2. Nuclei in the head of the animals were
stained by Hoechst-33342 (blue). B) Line profile plots of the fluorescence intensity of NLSCherry and Npp-1::GFP versus the distance in microns on wild-type (left) and deg-3(u662)
animals (right). Plots were obtained by scanning the fluorescence intensity along the dashed
black line in the inset. C) Confocal analysis of wt N2 animals overexpressing NGY (green)
and NLS-Cherry (red) driven by Punc119, respectively localized in the cytosol and in the
nucleus.
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