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Supplementary Figures
Supplementary Figure S1. Blockade of spontaneous Ca2+ activity in dissociated
thalamic neurons and downregulation of DCC protein in Kir-silenced neurons.
A. Dissociated thalamic cells transfected with Gfp or co-transfected with the Kir and Gfp
plasmids, and loaded with the Rhodamine Red calcium indicator (red). Ca2+ activity was
recorded in active cells in both Gfp-positive and Kir-positive cells (yellow). Quantification
of the active cells in both control and Kir-transfected cultures. **p<0.01; two-tailed
Student’s t-test. The data are presented as the means ± s.e.m. Scale bar, 50 µm.
B. Western blot showing the DCC protein in lysates from E13.5 thalamic neurons
transfected with Gfp or Kir and cultured for 60 hr. Quantification of the data shown (n =
3). *p<0.05, two-tailed Student’s t-test. ID, integrated density. The data are presented as
the means ± s.e.m.
C. Tuj1 immunostaining of dissociated thalamic cells after 60 hr in vitro revealed an
increase in axonal growth of chronically depolarized cells (in the presence of 2.5 mM of
KCl) when compared to the controls (5 mM control: 100 ± 3.9%, n = 8 coverslips from 3
independent experiments; 7.5 mM: 149 ± 3.4%, n = 8 coverslips from 3 independent
experiments). Nuclear counterstained by Dapi is shown in blue. Quantification of the
axonal length. ***p<0.001; Two-tailed Student’s t-test. The data are presented as the
means ± s.e.m. Scale bar, 100 µm.
D. Western blot showing the DCC protein in lysates of E13.5 thalamic neurons under
control conditions or in the presence of 2.5mM KCl to increase spontaneous activity for
60 hr. Quantification of the data shown (n = 3). **p<0.01, Two-tailed Student’s t-test. ID,
integrated density. The data are presented as the means ± s.e.m.
Supplementary Figure S2. Netrin-1-deficient thalamic axons are shorter.
A. In situ hybridization and immunocytochemistry for Netrin-1 in dissociated E13.5
thalamic neurons. Arrowheads indicate the expression of Netrin-1 in individual cells.
Nuclear counterstained by Dapi is shown in blue.
B. Quantification of the lactate dehydrogenase activity (LDH) in Netrin-1+/+ and Netrin-1–
/–
thalamic cultures to assess cell-death, showing that there are no significant change
between Netrin-1+/+ (n = 7) and Netrin-1–/– (n = 6) conditions. Two-tailed Student’s t-test.
Data are presented as mean ± s.e.m.
C. Tuj1 immunostaining revealed that thalamic axon growth decreased in the absence of
Netrin-1. Nuclear counterstained by Dapi is shown in blue. Scale bars, 100 µm lower
power panels and 50 µm higher power panels.
D. Quantification of the data shown in C and data not shown. **p<0.01 and ***p<0.001;
Kruskal-Wallis test with Dunn’s post hoc analysis. The data are presented as the means
± s.e.m.
Supplementary Figure S3. Conserved sites mutated in the Dcc promoter.
A. The ECR Browser was used to identify conserved sequence domains upstream of the
first Dcc exon (yellow and blue). The horizontal blue line depicted above the graph
indicates the start of intron1 and the arrow indicates the transcriptional orientation of the
Dcc gene. The yellow box corresponds to the 5’-UTR, while the blue box corresponds to
the coding sequence in the first exon. From top to bottom, a comparison of the human
genomic sequence of the dog, opossum, rat, mouse, macaque and chimpanzee is
shown, as indicated by the cartoon located on the right. The colored peaks (red:
intergenic regions; green: transposons and simple repeats; yellow: UTRs; blue: coding
exons; salmon: intronic regions) indicate regions ≥ 100 bp with an identity of at least
70%. The green box highlights a highly conserved peak 269 bp upstream of the 5’-UTR
start that contains the AP-1 binding site. The red box highlights a conserved peak
matching the 5’-UTR start and that contains the NF-kB site.
B. Multiple sequence alignments of a 60 bp region surrounding the two-conserved
potential transcription factor binding sites: asterisks indicate identity. The alignments
were obtained with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).