Key words: Intermediate filaments, nestin, Cdk5, muscle ...Nestin, a member of the cytoskeletal family of intermediate filaments, regulates the onset of myogenic differentiation through
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Figures
Figure 1 Nestin knockout mice weigh less and have less lean mass. (A) Body weight of
wildtype (WT) and nestin knockout (NesKO) male mice at 3 months (WT n=75; NesKO
n=68; p=0.0027 by Student´s t-test with Welch´s correction). NesKO mice were found to
weigh significantly less. (B) Total body weight mean values of WT and NesKO male mice at
age >15 months (range 15-17 months; WT n=9; NesKO n=11; p=0.0372 by Student´s t-test
with Welch´s correction. (C) Body composition of male mice at the age of 3 months. Absolute
values of lean (left panel) and fat mass (right panel) in grams are displayed in the scatter plots.
In comparison with WT mice (n=36), NesKO mice (n=33) had significantly less lean mass
(p=0.0048 by Student´s t-test with Welch´s correction). Average fat mass was not
significantly different (WT n=36; NesKO n=33). (D) Body composition measurement of WT
and NesKO male mice at age >15 months. Left panel: average lean mass (WT n=8; NesKO
n=8; p=0.0221 by Student´s t-test with Welch´s correction. Right panel: average fat mass
(WT, n=8; NesKO n=8; n.s.).
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Figure 2 NesKO skeletal muscle shows muscle type-specific weight differences and a
and extensor digitorum longus (EDL) muscles were isolated and weighed. While the Sol
weight did not show a marked difference, both TA and EDL muscles from nestin null animals
weighed significantly less (WT n=4; NesKO n=7; p=0.0401, and 0.0391, respectively, by
Student´s t-test with Welch´s correction). (B) The average myofiber size from Sol and EDL
histological sections , does not differ between genotypes. WT n=3, NesKO n=3. More than
250 myofibers from Sol and 500 myofibers from EDL were counted from each mouse. (C)
Myofiber size plotted as a frequency distribution for Sol (left panel) and EDL muscles (right
panel), respectively. (D) H&E stained TA, soleus and EDL muscles were investigated for
morphological aberrations. White arrowheads mark myofibers with centrally located nuclei
(CLN), denoting the presence of degenerating/regenerating myofibers, the occurrence of
which tended to be higher in NesKO muscles. H&E images are 0.12 mm x 0.12 mm in size.
(E) The amount of myofibers with CLN was calculated from two muscle sections per mouse.
The average amount of CLN per mm2 was determined, demonstrating that nestin null mice
tend to have a higher incidence of CLN than WT mice. N represents the number of mice;
from each mouse only muscles from the left leg were used. The difference was statistically
significant in the TA muscle (p=0.0249, Mann-Whitney test).
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Figure 3 NesKO myoblast cultures have higher Cdk5 activity, but differentiate at a
comparable rate to the WT. (A) Myoblasts isolated from WT and NesKO mice were
immunolabelled for nestin to demonstrate the absence of nestin in NesKO myoblasts. WT
myoblasts exhibited a filamentous nestin labeling in the proliferating cultures. Scale bars=10
µM. (B) Differentiating WT and NesKO P3 myoblasts were analyzed by Western blotting for
expression of nestin and other IFs. No recurring differences in the expression levels of other
IFs could be detected. (C) Myoblasts were isolated from adult male mice, induced to
differentiate, and samples were collected for Western blotting at indicated time points,
representing the progress of differentiation. Myoblasts isolated from WT and NesKO mice
differentiate equally, as determined by the expression of desmin and troponin t. The protein
levels of p35 were, however, increased in NesKO myoblasts. (D) Cdk5 was
immunoprecipitated from myoblasts and subjected to activity measurements in vitro using
histone H1 as a substrate. The γ-32P ATP incorporation into histone was considerably
increased in nestin null myoblast cultures, denoting that Cdk5 activity is increased in NesKO
myoblasts. The isolation of myoblasts and all subsequent experiments were repeated three
times.
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Figure 4 Satellite cell expansion is affected in the absence of nestin. (A) Intact myofibers
were isolated from EDL muscle, and live fibers were fixed immediately. Myofibers were
stained for the satellite cell marker Pax7 and the number of Pax7+ cells was calculated.
Results are visualized as the fraction of Pax7+ cells over the total amount of myonuclei
(>2000 myonuclei counted per mouse; n=3+3; n.s.), scale bars=50 µM. White arrowheads
mark Pax7+ cells. (B) Myofibers isolated from WT and NesKO mice were cultured in
floating conditions for 72 hours. Myofibers with associated satellite cells were then fixed, and
immunolabeled with Pax7 and MyoD antibodies to assess the proportion of cells that have
committed to the myogenic lineage (MyoD+). No obvious difference in satellite cell
commitment was observed; scale bars=10 µM. (C) Single satellite cells emerging from
cultured myofibers were manually tracked with Fiji/TrackMate, and the time between satellite
cell divisions was calculated (WT 9.52 hours n=111, and NesKO 10.6 hours n=111, p<0.0001
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by Student´s t-test with Welch´s correction). (D) Isolated myofibers were plated on Matrigel-
coated dishes, and emerging satellite cells were imaged continuously with Cell-IQ-imaging
platform. The numbers of proliferating satellite cells was calculated at indicated time points
with Cell-IQ Analyser software. The average number of cells per field at 83 hours was 219 for
WT and 178 for NesKO; p=0.0054 by Student´s t-test, n=3).
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Figure 5 Muscle regeneration after injury is delayed in NesKO mice. (A) TA muscles of
WT mice were injured and samples were collected for paraffin sections after indicated time
points (days after injury). Samples were immunolabeled with nestin antibody, demonstrating
the kinetics and localization of nestin expression in healing muscle. Black arrowheads mark
single-nucleated myoblasts. White arrowheads mark multinucleated myotubes. Upper images
are 1 mm x 1 mm and inserts below 0.2 mm x 0.2 mm in size. (B) WT (n=5) and NesKO (n=5)
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mice were subjected to muscle injury, and the wound was allowed to heal for 28 days. The
regenerating muscle area (defined by the presence of CLN in myofibers) was calculated and
compared to the total muscle area, revealing that NesKO mice have a significantly larger area
occupied by regenerating myofibers 28 days after injury (WT=16.10% vs. NesKO=26.06%;
p=0.0023 Student´s t-test). The injured area is marked with asterisk and is outlined in red.
Whole muscle sections show representative quantification results of the injured muscle area.
(C) H&E-stained samples from the injured muscles were inspected for histological aberrations,
but showed no obvious difference, except in the area occupied by regenerating fibers. Images
are magnified inserts from the muscle sections in panel B. H&E images are 0.3 mm x 0.3 mm
in size. (D) Fibrotic deposits were studied after injury with Picrosirius red stain against
collagen, imaged with bright field microscope, after which images were transformed to binary
black-and-white images to enhance the visibility of the collagen extracellular matrix
(visualized in black). The images are 0.5 mm x 0.5 mm in size.
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A
B
Figure S1 Voluntary running activity and food consumption of the animals. Related to figure 1. (A) 8 WT and 8
NesKO mice aged 3 months were allowed to run for 9 days on wireless running wheels (Med Associates Inc.) in
individual cages and the number of laps for each mouse was recorded with Wheel Manager software (Med Associates
Inc.). The results are presented as total number of laps per 24 hours for each mouse. We could not observe changed
running behavior between genotypes. (B) The average food intake of male mice at the age of 1 and 2 months was
followed for 5 days, with no obvious genotypic alterations.
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information
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A B
Figure S2 Weight and body composition alterations of adult female NesKO mice. Related to figure 1. (A) Total body weight
average values of WT and NesKO female mice at the age of 3 months (WT n=32; NesKO n=32; p=0.0038 by Student´s t-test with
Welch´s correction). (B) EchoMRI body composition measurement of WT and NesKO female mice at age of 3 months. Absolute
mean values of lean and fat mass are displayed as mean±s.e.m. Lean mass: WT n=32; NesKO n=32, p < 0.0001 by Student´s t-test
with Welch´s correction. Fat mass: WT n=32, NesKO n=32, n.s.
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information
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Day 14Day 7
Nestin
NesKO
Figure S3 Absence of nestin immunoreactivity in regenerating NesKO muscle. Related to figure 5. Nestin null
muscle was immunolabeled with nestin antibody 7 and 14 days after injury to control antibody specificity, when nestin
immunoreactivity was strongest in the wound area (white arrowheads mark multinucleated myotubes) in the
corresponding WT samples presented in Figure 5A. Upper images are 1 mm x 1 mm and inserts 0.2 mm x 0.2 mm in
size.
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information
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0 h
40 h
Myofiber explant cultures
Live imaging
Manual lineage tracking
Cell numberquantification
Cell doubling time (Fig. 3C)
Proliferation curve(Fig. 3D)
A
B
C
Figure S4 Workflow for live myofiber imaging. (A) After isolation, myofibers were allowed to attach to the substrate for 48
hours, and then imaged continuously with phase contrast microscopy (Cell-IQ) (Supplementary video 1). The acquired data was
processed with Cell-IQ Analyser to quantify the number of cells in the immediate vicinity of myofibers during several time points.
Alternatively, the proliferative behavior of satellite cells was studied by manual lineage tracking (Fiji/TrackMate). The time
between two cell divisions was extracted from the lineage trees as a measure of cell doubling time. (B) Representative images of
myofiber explant cultures from live cell imaging at initiation of imaging (0 hours) and at 40 hours. At 40 hours, satellite cells have
migrated out from the fibers and proliferated. (C) An example of a cell lineage tree generated by TrackMate.
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information
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Supplementary table 1 Antibodies used in the study
Antibody Clone/cat. number Source Used for Dilution
Actin AC-40 Sigma-Aldrich WB 1:1000
Cdk5 DC-34 Life Technologies WB, IP 1:1000
Cdk5 C-8 Santa Cruz WB 1:200
Desmin #4024 Cell Signaling WB 1:1000
GAPDH 14C10 Cell Signaling WB 1:2000
Hsc70 SPA-810 Stressgen WB 1:2000
MyoD M-318 Santa Cruz ICC 1:100
Nestin 556309 BD Pharmingen IHC, ICC 1:200
Nestin 611659 BD Pharmingen WB 1:1000
p35/p25 C-19 Santa Cruz WB 1:200
Pax7a AB_528428 DSHB ICC 1:20
Syneminb Prof. Omar Skalli WB 1:100
Troponin t JLT-12 Sigma-Aldrich WB 1:200
Vimentin 550513 BD Pharmingen WB 1:1000 aThe Pax 7 antibody developed by A. Kawakami, Tokyo Institute of Technology, was obtained from the Developmental Studies Hybridoma Bank, Created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA. b The synemin antibody was a kind gift from Professor Omar Skalli, University of Memphis.
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information
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Movie S1. Live imaging of myofiber explant cultures
J. Cell Sci. 130: doi:10.1242/jcs.202226: Supplementary information