Fig 2: Nup133 N- and C-terminal protein fragments bind nucleoporins differentially. (A) Full length human Nup133 (1141 aa) and specific mapped domains are shown. The N-terminal, designated Nt-Nup133, consisted of 805 aa and contained the β-propeller domain, the ALPS motif, and a portion of the α-helical domain. The C-terminal Ct-Nup133 construct consisted of aa 917–1141 and contained the site for Nup107 interaction (aa 934–980). (B–C) U20S 2–6–3 cells were transfected with the designated Nup133 fragment. The extent of nucleoporin recruitment was then tested by staining with anti-LacI antibody together with antibodies to Nup160 (B-C) or Pom121, ELYS, Nup53, Nup98, and Nup214 (D). The percentage of transfected cells showing rim localization is shown in last column of (D). The comparative data for full length Nup133 is presented in Table 1.

Fig 3: Lrrk2-deficiency caused nuclear hypertrophy and increased nuclear invaginations in SPNs after prolonged culture. a Co-staining of Lamin B and CTIP2 in Lrrk2+/+ and Lrrk2−/− SPNs after 3 weeks in culture. Scale bar, 10 μm. b, c The areas of SPN nuclei were measured from six independent Lrrk2+/+ and Lrrk2−/− cultures (b) and cumulative (Cum.) frequency was calculated to show the nuclear size distribution in each genotype (c). N = 300 neurons per genotype. Conditional logistic regression test, ****p < 0.0001. d Ratio of SPN nuclei containing ≥1 invagination was calculated from three independent Lrrk2+/+ and Lrrk2−/− cultures. N = 200 neurons per genotype. Unpaired t-test, *p = 0.0181. e, f Sample EM images of cultured Lrrk2+/+ and Lrrk2−/− striatal neurons (e). The boxed area was shown in f. M indicates mitochondria. Scale bars: 2 μm (e), 0.5 μm (f). g-j The nuclear area (g), perimeter (h), and circularity (i), as well as the ratio of nuclei containing 0 to 4 invaginations (j) were calculated from the EM images. N = 7 and 15 neurons for Lrrk2+/+ and Lrrk2−/− cultures. Unpaired t-test, **p = 0.0018 (area), **p = 0.0045 (perimeter), ***p = 0.0005 (circularity). k Co-staining of Lamin B and dsRed-Mito in a Lrrk2−/− striatal neuron. Scale bar, 5 μm. l Co-staining of NUP98 and Lamin B in a Lrrk2−/− striatal neuron. Scale bar, 5 μm

Fig 4: Distinct localization of two groups of NUP98-regulated developmental genes.(A) Criteria for counting gene localization as ‘Periphery’ or ‘Non-Periphery’, with LMNB staining in green and FISH signal in red. Genes counted as ‘Periphery’ were localized within 0.5 µm of the nuclear lamina. (B) Percentage of periphery localization of NUP98 binding genes through development, i.e. in ESC (yellow), NeuPC (blue) and Neuron (purple), determined from IF-FISH experiments. Error bars were calculated as standard deviation from triplicates for a total of at least 100 cells using 3D reconstruction of images. (C, D) Representative 3D IF-FISH images showing the localization of (C) group I genes (GRIK1) and (D) group II genes (GPM6B) through development, i.e. in ESC, NeuPC, and Neuron. FISH probes were shown in red, LMNB staining in green, and Hoechst in blue. Each set of images includes the x-y, y-z and x-z planes that cross at the FISH probe signal. (E) Model of two groups of NUP98-gene interaction. Group I genes are at the beginning stage of developmental induction in neural progenitor cells and interact with NUP98 at the nuclear pores in the NE, and subsequently translocate to intranuclear sites upon full induction in neurons. In contrast, group II genes are already greatly activated in NeuPCs and interact with NUP98 at intranuclear sites away from the NE. The percentage of genes observed at the nuclear periphery at each stage was indicated.

Fig 5: Stimulation with NMO IgG induces canonical NFκB signaling. a Lysates (50 μg per lane) from astroglial cells without stimulation (UNT), after 20-min stimulation with 20 ng/mL TNFα (TNF), as a positive control, or after stimulation with 100 μg/mL NMO IgG (NMO) for 30, 60, or 120 min were probed by immunoblot with antibodies specific for either phosphorylated IκB-α (pIκB-α) or total IκB-α. For each antibody, panels are from the same membrane and exposure. b Lysates (50 μg per lane) from unstimulated cells (UNT) or following stimulation for 5, 15, 30, 60, or 120 min with 100 μg/mL NMO IgG (NMO) or control IgG (CON) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. Accumulation of pIκB-α protein was evident after stimulation with NMO IgG but not in response to the control IgG. This panel is representative of 2 independent experiments. c Cells grown on glass coverslips were probed by immunofluorescence using antibodies against the astrocyte marker glial fibrillary acidic protein (GFAP) (red) and NFκB p65 (green) after stimulation for 20 min with 20 ng/mL TNFα or stimulation for 60 min with 100 μg/mL NMO IgG (NMO) or CON IgG (CON). In some experiments, NFκB pathway inhibitors were added to the cells for 2 h prior to stimulation. Robust nuclear translocation of p65 was observed following stimulation with NMO IgG and this translocation was blocked when cells were pretreated with NFκB inhibitors. Inhibitor concentrations: 50 μM MG132 (MG), 30 μM BAY 11–7082 (BAY), 30 μM SN50, 30 μM NF-kappa-B essential modulator (NEMO) binding peptide (NBP). These panels are representative of at least 2 independent experiments performed in duplicate. DAPI-labeled nuclei are shown in blue. Scale bar: 50 μM. d Cellular homogenates were fractionated into nuclear and cytoplasmic components prior to the preparation of lysates. Fractions (10 μg per lane) from unstimulated cells (UNT), cells stimulated for 20 min with 20 ng/mL TNFα (TNF), and cells stimulated for 60 min with 100 μg/mL NMO IgG (NMO) or CON IgG (CON) were probed by immunoblot for levels of p65. The relative purity of the fractions was determined by immunoblotting for the nuclear marker NUP98 and the cytoplasmic marker α-tubulin. Increased nuclear p65 protein was observed following stimulation with NMO IgG as compared to UNT and CON IgG-stimulated samples, consistent with the immunofluorescence in c. This blot is representative of 3 independent experiments. e Lysates (50 μg per lane) were probed by immunoblot with antibodies for either pIκB-α or total IκB-α following no stimulation (UNT), after 60-min stimulation with 100 μg/mL of CON IgG or NMO IgG, or after stimulation with NMO IgG following pretreatment with NFκB pathway inhibitors. BAY, SN50, and NBP treatments reduced pIκB-α levels following NMO IgG stimulation to the levels observed in unstimulated and CON IgG-stimulated samples. MG132 treatment resulted in accumulation of pIκB-α due to proteasome inhibition, as expected. This blot is representative of at least 2 independent experiments