1. a GM
DM1
DM3
DM5
DM7 b
MHC
MHC +DAPI GM
DM4
Supplementary Figure S1. (a) Representative images of C2C12 during differentiation. C2C12 myoblasts proliferate in growth medium (GM) and differentiate to myotube in serum-depleted differentiation medium (DM) from 1 to 7 days. (b) Immunostaining of MHC in C2C12 cells cultured in GM and in DM for 4 days.

2. Day 0
Day 1
Day 3
Day 5
Day 10
Supplementary Figure S2. H & E images of tibialis anterior muscle cross sections in mice on days 0, 1, 3, 5 and 10 post-CTX injury. Evolution of the morphological changes were seen in the diaphragm of C57BL/6 mice with muscle regeneration, leading to accumulation of mononucleated cells and their differentiation to form new myofibers to repair damaged muscle. Arrows indicate the regenerating myofiber (Day 5).

3. b a
Day 1
Day 3
Day 5
Day 10
Day 0
Supplementary Figure S3. Expression of Pax7 (a) and MyoD (b) mRNAs in tibialis anterior muscles of C57BL/6 mice on days 0, 1, 3, 5 and 10 post-CTX injury. The error bars depict the mean ± standard deviations of the samples from six mice in each group.

4. a
EdU
DAPI
Merge
miR-NC
miR-17
miR-20a
miR-92a b
EdU
DAPI
Merge
dTuD-NC
dTuD-17
dTuD-20a
dTuD-92a
Supplementary Figure S4. The cell proliferation analysis was performed by EdU incorporation of C2C12 myoblasts infected with lentiviruses expressing miR-17, -20a or -92a (a) and miRNA inhibitors dTuDs targeting miR-17, -20a, or -92a (dTuD-17, -20a or -92a) or dTuD-NC (b).

5. a b C α-sarcomeric actin DAPI α-sarcomeric actin+DAPI miR-NC miR-mix
kDa
myogenin 35 55
β-tubulin
Supplementary Figure S5. (a) Immunostaining of α-sarcomeric actin
in mouse primary myoblasts in GM. (b) Western blot and (c) quantification of myogenin in the primary myoblasts transfected with a mixture of miRNA mimics (miR-17, -20a and -92a) or NC and transferred to DM for four days. Beta-tubulin served as the loading control.

6. a b C2C12 myogenesis Relative expression (/GM)
Skeletal muscle regeneration
Relative expression (/Day 0)
Supplementary Figure S6. (a) Expression of ENH1 protein and mRNAs (miR-17, -20a and -92a) during C2C12 myogenesis. The error bars depict the mean ± standard deviations of three independent cell samples. (b) Expression of ENH1 protein and mRNAs (miR-17, -20a and -92a) during mouse skeletal muscle regeneration after CTX-injury. The error bars depict the mean ± standard deviations of three independent cell samples. The error bars depict the means ± standard deviations of the samples with six pigs in each group.

7. si-NC si-ENH1 OE-NC OE-ENH1
EdU
DAPI
Merge
si-NC
si-ENH1
OE-NC
OE-ENH1
Supplementary Figure S7. The cell proliferation analysis was performed by EdU incorporation of C2C12 myoblasts transfected by si-ENH1 or plasmids expressing ENH1.

8. Supplementary Figure S8
Supplementary Figure S8. Expression of Id1, Id2, Id3 and Id4 mRNAs in myoblasts. The error bars depict the mean ± standard deviations of three independent cell samples. GAPDH used as the mRNA endogenous control.

9. Skeletal muscle regeneration
C2C12 myogenesis
Protein expression b
Day 0
Day1
Day3
Day 5
Day 10
kDa
M1 M M M M1 M2 M1 M2 M1 M2 15
Id1 55
Tubulin c
Skeletal muscle regeneration
Protein expression
Supplementary Figure S9. (a) Protein expression of ENH1 and Id1 during C2C12 myogenesis. The error bars depict the mean ± standard deviations of three independent cell samples. (b) Western blot analysis of Id1 protein expression during skeletal muscle regeneration after CTX-injury. M1 and M2, mouse 1 and mouse 2. (c) Protein expression of ENH1 and Id1 during skeletal muscle regeneration. The error bars depict the means ± standard deviations of the samples with six mice.

10. Ad-miR-NC Ad-miR-20a Day 0 Day 1 Day 3
Supplementary Figure S10. H & E images of the tibialis anterior muscle cross sections of mice injected with Ad-miR-NC or Ad-miR-20a on 0, 1 and 3 days post-CTX injury. Scale bar, 200 μm.

11. Supplementary Figure S11. miR-17-92 promoter luciferase assays
Supplementary Figure S11. miR promoter luciferase assays. C2C12 myoblasts were co-transfected with the vectors expressing E2F1 and the miR promoter luciferase reporter containing E2F binding sites (Wt) or mutant E2F sites (Mut). The error bars depict the means ± standard deviations of three measurements. ***P <