Legends to Supplementary Figures
Supplementary Figure 1. Representative video image sequences of injured Lgals1-/- mice treated
with vehicle. (a) Yellow arrows show spinal column and tail position. (b) Representative images of
a mouse on a bridge-shaped metal grid. (c) Yellow lines in the left image indicate placement of
paw, the right image shows the tail position.
Supplementary Figure 2. Representative video image sequences of injured Lgals1-/- mice treated
with 1 µg/µl Gal-1. (a) Green arrows show two complete cycles of hindlimb movement. The yellow
arrow in the same image sequence shows tail position. (b) Representative images of a mouse on a
bridge-shaped metal grid (yellow arrow show the tail position). (c) Yellow lines in the left picture
show the placement of the paw, right picture shows tail position.
Supplementary Figure 3. Representative video image sequence of sham Lgals1-/- mice. (a)
Representative images of a mouse show movement of hindlimb and tail position. Green arrows
indicate a complete cycle of hindlimb movement. (b) This animal was used to setup the gridwalking apparatus test and showed capacity to cross the bridge-shaped metal grid using hindlimb
force and balancing with its tail. (c) Yellow lines in the left picture show the placement of the paw
to setup analysis cut-off. Right picture shows tail position.
Supplementary Figure 4. Motoneurons downstream of the lesion site. (a) Bar graph shows
quantification of gray matter area occupied by motoneurons downstream of the lesion site. Values
represent the mean ± s.d. of 5 independent experiments (n=3 mice per group). (b-d) Triple staining
with III-tubulin, GFAP and DAPI. (b) The right inset represents a segment of the lesion site in
vehicle-treated WT mice. The figure is a magnification of the dotted white frame in the inset, which
shows gray matter downstream of lesion site. The 20X magnification below (dotted frame 1) shows
an area occupied by motoneurons (asterisk). (c) Figure shows the downstream area of vehicletreated Lgals1-/- mice, which is highlighted in the free floating right inset (black arrow). (d) The
figure shows gray matter in Lgals1-/- mice treated with 1 µg/µl Gal-1. The 20X magnification below
(dotted frame 1) shows an area occupied by motoneurons in the downstream area. Scale bars: 250
µm for 10X. 150 µm for 20X.
Supplementary Figure 5. Localization of PlexinA4 in a representative neuron from an Lgals1-/mouse treated with Gal-1. The left picture displayed a triple staining with III-tubulin, PlexinA4
and Gal-1 followed by confocal microscopy of a neuron at the epicenter of the lesion site. The right
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picture shows a computer-generated 3-D image of PlexinA4 (green) and Gal-1 (red) clusters
localized in the neuron body (dotted black frame in the left image). The panel below shows a
quantitative correlation analysis using Mander’s algorithm as well as a quantitative colocalization
rate analysis. Data are representative of 4 independent experiments (n=3 mice per group). Scale bar,
20 µm.
Supplementary Figure 6. Locomotor recovery induced by M-Gal-1 treatment. (a) Quantitative
BBB score analysis is shown in the bar graph. Values represent the mean ± s.d. of 5 independent
experiments (n=3 mice per group). ***P < 0.001, ns = not significant, using one-way ANOVA
followed by Tukey´s tests. (b-c) Representative video image sequences of Lgals1-/- mice treated
with 1 µg/µl M-Gal-1 as compared with vehicle-treated WT mice. Green arrows show hindlimb and
column position for each image in both conditions. (d–e) Representative images of a mouse on a
bridge-shaped metal grid (yellow, green and red arrows show the tail, the paw and the column
position, respectively).
Supplementary Figure 7. Spinal cord scar size after M-Gal-1 treatment. In all representative
images, the left side is cranial. Pictures show scar localization delimited by white dotted line. Also,
in the bottom left corner of each picture, a spectrum graph, emphasizes higher signal in red and
lower signal in blue. Calibration is shown on the right band (0–255 colors). Quantitative analysis is
shown in bar graphs below. Values represent the mean ± s.d. of 5 independent experiments (n=3
mice per group). ***P < 0.001, **P < 0.01 using one-way ANOVA followed by Tukey´s tests.
Supplementary Figure 8. Mechanism of axonal regeneration induced by Gal-1 treatment: a model.
(a) Scheme of NRP-1 structure. The N-terminal extracellular domain contains 835 amino acids, the
transmembrane domain contains 23 amino acids and the cytoplasmatic domain contains 44 amino
acids. The extracellular region is divided into three domains formed by a1⁄a2 (CUB) domain, b1⁄b2
domain and c domain. The cytoplasmatic domain has a PDZ-domain binding the NRP-interacting
protein. (b) The scheme shows the extracellular target of Sema3A-binding site which is produced
after SCI. (c) The scheme shows blockade of the Sema3A-NRP-1 complex by Gal-1 when
presented in its dimeric form.
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