Resultados de investigación

Predictive clinical outcomes and biomarkers at T1 of change maximum heart rate., Peer reviewed

Shown is α-DG glycosylation status in Poglut1-cKO and control mouse muscles at P0 and P20 assessed by western blotting using antibodies against the glycosylated form of α-DG (αDG-IIH6) and the core α-DG, and by laminin overlay assay. An antibody against β-DG was used to assess loading. Wheat germ agglutinin-enriched muscle lysates from control and cKO mice were used. The western blot showed a reduced expression level of glycosylated α-dystroglycan in cKO mice at P0 and P20, as well as a diminished binding activity in the laminin overlay assay when compared to control mice. Each lane represents an individual animal. Boxes mark samples taken from non-adjacent wells of the same gel., Peer reviewed

Shown is the DAPI, laminin co-staining of TA muscles from the indicated genotypes at E16, P0, and P21. Note a higher frequency of myofibers with internal nuclei in P0 and P21 Poglut1-cKO muscles compared to sibling controls without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+). Quantification is shown in Fig 3A., Peer reviewed

Shown is the PAX7 MYOD Ki67 triple staining of myogenic progenitors isolated from forelimb and hindlimb muscles of P4 Poglut1-cKO and sibling controls without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) and cultured in proliferation media for five days. TOPRO-3 marks the nuclei. Yellow arrowheads, white arrows and white arrowheads mark examples of quiescent muscle stem cells (PAX7+ MYOD– Ki67–), cycling progenitor cells (PAX7+ MYOD+ Ki67+), and non-cycling precursor cells (PAX7– MYOD+ Ki67–), respectively. Quantifications are shown in Fig 3H., Peer reviewed

Representative MS/MS spectra are shown on the left, and Extracted Ion Chromatograms (EICs) on the right for peptides containing the POGLUT1 consensus site from mouse NOTCH3 EGF repeats. A) Peptide with O-glucose monosaccharide on EGF2. B) Peptide with O-glucose-xylose-xylose trisaccharide on EGF3. C) Peptide with O-glucose-xylose-xylose trisaccharide on EGF9. D) Peptide with O-glucose-xylose-xylose trisaccharide on EGF11. E) Peptide with O-glucose-xylose-xylose trisaccharide on EGF13. F) Peptide with O-glucose-xylose-xylose trisaccharide and O-GlcNAc monosaccharide on EGF15. G) Peptide with O-glucose-xylose-xylose trisaccharide and O-GlcNAc monosaccharide on EGF16. H) Peptide with O-glucose-xylose-xylose trisaccharide and O-fucose-HexNAc-Hexose trisaccharide on EGF17. I) Peptide with O-glucose-xylose-xylose trisaccharide and O-fucose monosaccharide on EGF19. J) Peptide with O-glucose-xylose-xylose trisaccharide and O-fucose monosaccharide on EGF23. K) Peptide with O-glucose monosaccharide on EGF25. L) Peptide with O-glucose-xylose-xylose trisaccharide and O-fucose monosaccharide on EGF29. M) Peptide with O-glucose-xylose-xylose trisaccharide on EGF32. The b- and y-ions are indicated in blue and red font, respectively. ~ indicates ions that lost glucose in the gas phase during fragmentation. Due to the lability of O-glycans in the gas phase during collision, these modifications can fall off which can lead to incorrect annotation of the modified amino acid. However, we can predict the correct position based on the putative consensus sequence for modification by POGLUT1., Peer reviewed

Raw numerical values and summary statistics for graphs shown in the paper., Peer reviewed

A) Images of Poglut1-cKO mouse (arrows) and their littermate controls at P0, P7, P14 and P21. B, C) Western blot images and quantification of relative levels of POGLUT1 from isolated satellite cell lysates (B, left panel) and whole muscle lysates (B, right panel) from forelimb and hindlimb muscles of Poglut1-cKO and sibling controls. Anti-GAPDH and anti-tubulin blots are shown as loading controls. Each western blot lane and each circle in the graphs represents an individual animal. D) TA muscle weights, tibia lengths, and TA muscle weights normalized by tibia length of each mouse. E) Survival curves of Poglut1-cKO and sibling control mice with regular diet and with soft food. n = number of animals. F–H) Body weights (F), open field test (G) and grip test (H) of control and cKO mice, with each dot representing an individual animal. I) Representative en face views of NMJs from the LAL muscle stained with BTX-Rho (red), which binds specifically to postsynaptic AChRs, anti-VAChT (green), which labels synaptic vesicles; and anti-neurofilament, which labels motor neuron axons (blue). Terminals of control and cKO mice at P20 are shown. J) Quantification of the postsynaptic areas in cKO and control terminals, in ages between P20 and P50 (Poglut1-Ctr: 5 mice (50 terminals); Poglut1-cKO: 5 mice (47 terminals)). Siblings without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) were used as controls in all experiments. Mean±SD. Two-way ANOVA with Šidák’s multiple comparisons test (F–H); unpaired t test (B, D, J). Row factor (the overall effect of genotype) is shown for F–H. ns: not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001., Peer reviewed

A) Images of TA muscle sections from mice harvested at E16, P0, and P21and stained for DAPI (blue), Laminin (green), and PAX7 (red). Asterisks mark myofibers separated by very weak Laminin staining. B) Quantification of PAX7+ cells per mm2 in sections from TA muscles harvested at E16, P0, and P21 from the indicated genotypes. C) Pax7 mRNA levels measured by qRT-PCR from limb muscles of control and cKO mice. D) Pax7 mRNA levels measured by qRT-PCR from satellite cells (SC) and other cells (non-SC) isolated from muscles of P4 control and cKO mice. E) Images of muscle sections from mice harvested at E16 (hindlimb), P0 (hindlimb), and P21 (TA) and stained for DAPI (blue), Laminin (green), and Collagen VI (red). Asterisks mark myofibers separated by very weak Laminin/ColVI staining. F–G) Representative images of M-Cadherin and PAX7 staining of TA muscle sections (F) along with the quantification of single/double positive cells per mm2 (G). In B–D and G, each dot represents an animal. Siblings without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) were used as controls in all experiments. Mean±SD is shown. Unpaired t test (B, C, G); one-way ANOVA with Tukey’s multiple comparisons tests (D). ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001., Peer reviewed

A) Quantification of percentage of myofibers with internal nuclei in TA muscles from the indicated genotypes at E16, P0, and P21. B) Violin plots of myofiber cross-sectional area (CSA) from P0 and P21 mice of the indicated genotypes. Horizontal lines indicate median ± one quartile. C) Immunofluorescence image of isolated myofibers of P21 mice stained with DAPI (blue) and Phalloidin (green) with quantification of the number of nuclei per μm of fiber length. D) Immunofluorescence images of TA cross-sections stained for DAPI (blue), eMHC (red), and Laminin (green), with quantification of eMHC+ fibers per mm2 of section area. E) Immunofluorescence images of myoblasts stained for PAX7 (green) and the nuclei (TOPRO-3, blue). F) Quantification of primary myoblasts from control and cKO mice (proliferation rate) at 1, 3 and 5 days of culture in proliferation medium. Note that the same number of cells was plated at the start of each culture to ensure comparability. G) Percentage of PAX7+ cells at 1, 3 and 5 days of proliferation. H) Pie charts showing the distribution of the myogenic populations based on their PAX7 and MYOD expression and cycling state (tracked by Ki67) on 1, 3, and 5 days of culture in proliferation medium. I) Immunofluorescence images of myoblasts stained for desmin (red) and myogenin (green) after three days of culture in differentiation medium. Nuclei were counterstained with TOPRO-3 (blue). J) Fusion index for cultures from panel I, defined as the percentage of nuclei in myotubes relative to the total number of nuclei in myogenic cells. K) Quantification of myogenin expression from indicated genotypes. In all graphs, each dot represents data from one animal. Siblings without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) were used as controls in all experiments. Mean±SD. One-way ANOVA with Tukey’s multiple comparisons test (F, G, H) or unpaired t test (A–D, J, K). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001., Peer reviewed

A) P21 mouse TA muscles were injected with CTX and harvested either 5 or 14 days post injection. Immunofluorescent staining [DAPI (blue), Laminin (green), and eMHC (red)] and H&E images of injured and non-injured TA muscles of control and mutant animals are shown. B) Staining of isolated myofibers from P21 mice with DAPI (blue) and PAX7 (red) and quantification of PAX7+ cells per myofiber. C) Staining of isolated fibers after 48 hours of culture for DAPI (blue), PAX7 (red), and MYOD (green). The arrow in the inset of the cKO panel marks a MYOD+ single cell occasionally seen in the cKO myofibers at this time. D) Staining of isolated fibers after 48 hours of culture for DAPI (blue) and Ki67(red). E) Quantification of clusters present in myofibers cultured for 48 hours post isolation. Siblings without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) were used as controls in all experiments. In B and E, each dot represents an animal, mean±SD, unpaired t test. ****P < 0.0001., Peer reviewed