Supplemental Figure Legends
Supplemental Figure S1. Table and hierarchal cluster of dysregulated microRNA
unaffected and sapje zebrafish at 5 and 30 dpf.
A. Clustal hierarchical analysis of
microRNAs dysregulated in unaffected (wild type and sapje heterozygotes) and affected
sapje (dystrophin-deficient) zebrafish at 5 dpf. B. The hierarchal clustering of 30 dpf
unaffected and dystrophic sapje fish. C. Real time qPCR of miR-199a-3p (miR-199a*)
during normal and DMD myogenic differentiation. Values normalized to 50% confluency
normal control and U6 snRNA expression. D. Evolutionary sequence alignments of
miR-199a-5p and miR-199b-5p sequences in vertebrates show a high degree of
sequence conservation. The 7-nt seed site is underlined and the C/U variant between
the sequences is indicated in bold. E. Real time qPCR of the human miR-199 stem
loop sequences demonstrating that miR-199a-2 is the most overexpressed transcript
induced in DMD muscle biopsies as compared to miR-199a-1, and miR-199b. Samples
normalized to unaffected human muscle and RNU6-2 loading controls.
F.
Altered
expression of dynamin transcripts (DNM1/2/3) in DMD muscle biopsies as compared to
normal muscle controls. Real time qPCR values normalized to 18sRib and normal
muscle expression.
* p-value < 0.005 ** p-value < 0.05. All samples analyzed in
triplicate in 3 separate normal or DMD cell lines.
Supplemental Figure S2. Evolutionary conservation of miR-199a-5p sites in
3’UTRs of WNT-signaling pathway components. MicroRNA-199a-5p binding sites of
human, mouse, and zebrafish WNT pathway genes (JAG1, WNT2, GSK3β, FZD4, and
1
CAV1) aligned with miR-199a-5p mature miRNA sequence. The 7-nt seed sight is in
bold letters.
Supplemental Figure S3. YY1 overexpression has a modest inhibitory effect on
miR-199-5p expression levels. A. Overexpression of YY1 inhibits the expression of
the 2kb hDNM3OS luciferase reporter construct in HEK293T cells. B. and C.
Overexpression of YY1 has little effect on miR-199a-5p transcript levels compared to
empty vector controls. Real time qPCR of miR-199a-5p levels in normal human primary
myoblasts (B.) and myotubes (C.) normalized to U6 snRNA. * p-value < 0.005 NS =
not significant.
Supplemental Figure S4. List of real time qPCR and ChIP primers and EMSA
oligos used in this manuscript.
manuscript. B.
A.
All real time qPCR primers used in this
ChIP primers for human genomic SRF sites found upstream of the
miR-199a-5p/miR-214 (DNM3OS) locus. C. EMSA oligo sequences containing the wild
type (endogenous) CArG boxes along with their corresponding 3-nucleotide mutated
(Mut) sequences.
Supplemental Figure S5. List of primary antibodies used in this manuscript. List
of primary antisera directed against the listed proteins used in this manuscript along
with manufacturer, and antisera dilution depending on assay. IF, immunofluorescence;
WB, western blot, ChIP, chromatin immunoprecipitation.
2
Supplemental Figure S6. Uncropped image scans of western blots used in this
manuscript. Uncropped image scans of all western blots shown in this manuscript
matched to the corresponding figure. Inset box highlights region used for figures in the
manuscript.
3