Article Figures & Data
Figures
- Figure S1. Nup210 is dispensable for muscle growth and survival.
(A) Total protein was isolated from control and Nup210 knockout quad muscle and Nup210 levels were analyzed by Western blots. Hsp90 was used as loading control. (B) Weight measurements of Nup210+/+ and Nup210−/− mice (n = 14–15) were taken every 2 wk. (C) Kaplan–Meier plot of Nup210+/+ and Nup210−/− mice (n = 19–20) measuring survival for up to 25 mo. (D) Fresh tissue weights for Quad and TA muscles isolated from Nup210+/+ and Nup210−/− mice (n = 3). Data are plotted as mean ± SEM.
- Figure 1. Nup210 is dispensable for mouse development.
(A) Schematic illustration of the generation of the Nup210 knockout (Nup210−/−) mouse from the HprtCre-mediated recombination of the loxP flanked exon 2 of Nup210 (Nup210f/f). (B) Nup210 was immunoprecipitated from quadricep (Quad), tibialis anterior (TA), and gastrocnemius (GAS) muscles and the amount of protein in the immunoprecipitated was analyzed by Western blot. Hsp90 in the input is used as loading control for the immunoprecipitation and IgG shows the levels of anti-Nup210 used. Asterisk shows oligomeric forms of Nup210 previously described (13). (C) Representative images of Nup210 immunofluorescence staining in TA muscle longitudinal sections obtained from Nup210+/+ and Nup210−/− mice. Scale bar, 25 μm. (D) Representative images of H&E full projections (top) and detailed views (bottom) of TA muscle transverse sections isolated from 6- to 8-wk-old Nup210+/+ and Nup210−/− mice. Scale bar 100 μm. (E) Representative images of immunofluorescence staining for Laminin in quadricep muscles from 6- to 8-wk-old mice. Scale bar, 100 μm. (F) Quantification of myofiber cross-sectional area distribution in quadricep muscles from 6- to 8-wk-old mice (n = 3). Data are binned in 250 μm2 bins and plotted as mean.
- Figure 2. Primary muscle satellite cells from Nup210 knockout mice efficiently differentiate into myotubes.
(A) Schematic illustration (top) and representative images immunofluorescence staining (bottom) of sorted satellite cells from Nup210+/+ and Nup210−/− mice after induction of myogenic differentiation. Arrows indicate undifferentiated muscle progenitor cells which lack Nup210 expression. Scale bar, 50 μm. (B) Representative immunofluorescence images of differentiated satellite cells from Nup210+/+ and Nup210−/− mice stained for myosin heavy chain (MHC) and Hoechst. Scale bar, 300 μm. (C) Nup210+/+ and Nup210−/− satellite cells were sorted, differentiated, and stained for MHC. The percentage of nuclei in MHC-positive cells (differentiation index) was quantified. (D) Nup210+/+/Pax7-CreERT2 and Nup210f/f/Pax7-CreERT2 were treated with tamoxifen and satellite cells were isolated and differentiated in vitro. (C) The differentiation index (percentage of nuclei in MHC-positive cells) was quantified as in (C). (D, E) Representative immunofluorescence images of cells from (D) stained for Nup210 and MHC. Scale bar, 100 μm. Data are plotted as mean ± SD.
- Figure S2. Nup210 levels increase in regenerating muscle fibers.
Sections from uninjured and BaCl2 injured TA muscles were stained for Nup210 (green) and Hoechst (Blue). Scale bar, 25 μM.
- Figure 3. Nup210 ablation results in delayed muscle regeneration.
(A) Young (6–8 wk old) Nup210+/+ and Nup210−/− mice were subjected to BaCl2-induced muscle injury and muscle regeneration was analyzed 5 d later. Top: Schematic illustration of experimental approach. Bottom: Quantification of myofiber cross-sectional area distribution. n = 3, data are binned in 250 μm2 bins and are plotted as mean. (B) Nup210+/+/Pax7-CreERT2 and Nup210f/f/Pax7-CreERT2 mice carrying a Cre-inducible tdTomato reporter (R26Td) were treated with tamoxifen before being subjected to BaCl2 muscle injury. The tdTomato-positive myofibers were analyzed by immunofluorescence in muscle sections. Laminin was used as counterstain to detect muscle fibers. Top: Schematic representation of experimental approach. Bottom: Representative immunofluorescence images from TA sections. Scale bar, 100 μm. (C) Young Nup210+/+ and Nup210−/− mice were subjected to BaCl2-induced serial muscle injury (three injuries total), and muscle regeneration was analyzed 25 d after last injury. Top: Schematic illustration of experimental approach. Bottom: Quantification of myofiber cross-sectional area distribution. n = 3, data are binned in 250-μm2 bins and are plotted as mean.
- Figure 4. Nup210 deficiency in muscle results in increased number of centrally nucleated myofibers.
(A) Representative H&E images (left) and quantification of centrally nucleated myofibers (right) of quadricep muscle from 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 3–9). Arrows denote centrally nucleated fibers. Scale bar 50 μm. (B) Representative H&E images (left) and whole section quantification of centrally nucleated myofibers (right) of quadricep muscle from 6- to 9-mo-old Nup210f/f and Nup210f/f/Acta1-Cre mice (n = 4–5). Arrows denote centrally nucleated fibers. Scale bar 50 μm. (C) Quantification of quadricep myofiber cross-sectional area isolated from 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 3). Data are binned in 250 μm2 bins. (D) Quantification of myofiber cross-sectional area distribution in TA isolated from 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 4). (A, B) Data are binned in 250 μm2 bins. **, P ≤ 0.01 by two-way ANOVA with Sidak’s multiple comparisons test (A), and unpaired t tests in (B). Data are plotted as mean ± SEM in (A, B) and as mean in (C, D).
- Figure S3. Evaluation of Quad and TA muscle.
(A) Quantification of total fiber count for quadricep muscles from 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 3). (B) Quantification of centrally nucleated myofibers in TA muscle from Nup210+/+ and Nup210−/− mice (n = 5–9).
- Figure 5. Nup210 is required for the maintenance of normal skeletal muscle fiber type distribution.
(A) Representative immunofluorescence images showing Type I (red) and II (white) fibers in soleus of 6- to 9-mo-old Nup210+/+ and Nup210−/− mice. (B) Quantification of fiber type distribution in soleus of 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 5). (C) Quantification of total fiber count for soleus muscles from 6- to 9-mo-old mice (n = 5). (D, E, F) Quantification of myofiber cross-sectional area for all fibers (D), Type I fibers (E), and Type II (IIa, IIx, and IIb) fibers (F) in soleus of 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 5). (G) Nup210+/+ and Nup210−/− mice were subjected to treadmill exercise and the time to exhaustion was quantified. (H) Nup210+/+ and Nup210−/− mice were subjected to 4 wk of voluntary running. The average distance ran per day each week was quantified and plotted. Data are plotted as mean ± SEM in (B), (C, G, H) and mean in (D, E, F). **P ≤ 0.01 by two-way ANOVA with Sidak’s multiple comparisons test.
- Figure S4. Quantification of fiber type distribution in soleus.
Quantification of fiber type distribution in soleus of 6- to 9-mo-old Nup210+/+ and Nup210−/− mice (n = 5). *P ≤ 0.05 by two-way ANOVA with Sidak’s multiple comparisons test.
- Figure 6. Nup210 ablation affects the expression of cell adhesion and immune genes.
(A) Volcano plot of RNA-seq transcriptome data for Nup210 knockout quad muscle relative to control muscle. Significantly up- and down-regulated genes are reported as red and blue dots, respectively (q < 0.05). Genes not changing expression are represented as gray dots. (B) Ingenuity Pathway Analysis of genes deregulated in Nup210 knockout quad muscle (>1.5 fold, q < 0.05). The top 20 pathways are shown. Green dots mark cell adhesion/cytoskeletal-related pathways, red dots show immune-related pathways, yellow dots show pathways associated with both. (C) Upstream transcriptional regulators predicted to be altered in Nup210 knockout muscle by Ingenuity Pathway Analysis. Only pathways with at least 10 target genes affected were included in the analysis. The top 20 pathways are shown. Mef2C is shown in blue. (D) Expression levels for Mef2C muscle structural target genes from the RNAseq data from. Data are plotted as mean ± SEM.
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