Supplemental Table/Figure Legends

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Loreen Newman
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1 MiR-26a is required for skeletal muscle differentiation and regeneration in mice Bijan K. Dey, Jeffrey Gagan, Zhen Yan #, Anindya Dutta Supplemental Table/Figure Legends Suppl. Table 1: Effect of overexpression and inhibition of mir-26a on myoblast differentiation. C2C12 cells were transfected three times at 24 h intervals with mir-26a or GL2 in GM and the cells transferred to DM. Cells were stained for Myogenin at 32 hour or MHC at 60 hour. In another experiment, 2 O-methyl antisense oligonucleotide against GL2 or mir-26a were transfected as in (A) and stained for Myogenin and MHC at 32 and 60 hour respectively. Fraction of Myogenin- or MHC-positive cells was determined randomly from 10 fields and data are presented relative to the GL2 control (100%). Suppl. Table2: Sequences List of qrt-pcr primers used in this study. Sequences of qrt- PCR primers used in this study are shown. Suppl. Figure 1: (A) Cardiotoxin-induced muscle degeneration and regeneration in mouse TA muscle. H & E staining of Fresh frozen (OCT) TA muscle sections from control, Day 1, 3, 5, 7, 10 and 14 post CTX injection are shown. (B) MiR-26a is upregulated in the Pax7- positive cells during muscle regeneration. MiR-26a and Pax7 co-staining is shown on day 7 post CTX injection. Yellow or white arrows indicate mir-26a and Pax7-positive or negative cells, respectively, located outside the skeletal muscle fibers. 1

2 Suppl. Figure 2: Changes in cell cycle profile after transfection of mir-26a in C2C12 cells. Primary FACS results for Fig. 2D. Propidium iodide staining for DNA content and FACS analysis data are shown. Mean +/- standard deviation of three measurements. Suppl. Figure 3: MiR-26a promotes mouse primary myoblast differentiation. (A) Mouse primary myoblast cells transfected with GL2 or mir-26a as in Fig. 2C and qrt-pcr performed for Myogenin and MHC. The results were normalized to GAPDH in the same sample and then again to the level in GL2-transfected cells. Mean +/- standard deviation of three replicates. (B) 2 O-methyl antisense oligonucleotides against GL2 or mir-26a were transfected as in Fig. 2G and qrt-pcr performed for Myogenin and MHC and data presented as in (A). Suppl. Figure 4: Predicted mir-26a target sites on Smad1 and Smad4 3 UTR. The RNAhybrid algorithm predicted mir-26a (green) matching target sites (red) on Smad1 3 UTR (A-D) and Smad4 3 UTR (E, F). The location of the sites on the 3 UTRs and the stability of binding (mfe, minimum free energy) are shown. The point mutations created are shown as. Suppl. Figure 5: C2C12 cells stably expressing Smad1orf do not decrease Smad1 either in the DM or in GM. C2C12 cells stably expressing various constructs as indicated were held in GM or induced to differentiate by transferring to DM and held in DM for 2 days. (A) Smad1 protein detected by Western blotting and GAPDH served as a loading control. (B) Quantitation of indicated bands in (A) normalized to GAPDH and again to that in cells with empty vector in GM. The exogenous Smad1 is expressed at 3x the level of endogenous Smad1 in GM. Suppl. Figure 6: Knockdown of mir-26a by antagomir or TuD in the neonatal and adult muscles increases Ezh2 mrna level. (A) Ant26a increases Ezh2 mrna level. qrt-pcr of Ezh2 on hind leg skeletal muscle, normalized to GAPDH in the same sample and then again to 2

3 the level in controls. Mean +/-standard deviation of the sample from three mice. (B-D) Ezh2 mrna level after injection of TuD-26a but before cardiotoxin injury and during regeneration (D7 & 14). qrt-pcr was performed and data represented as in (A). Suppl. Figure 7: Ki-67-positive cells in the Ant26a injected neonatal skeletal muscles coexpress Pax7. Confocal microscopy images of Ant26a injected neonatal skeletal muscles from Fig. 5D immunostained with Pax7 and Ki-67. Arrows in yellow and white indicate presence or absence of Pax7 in the ki-67-positive cells respectively. Suppl. Figure 8: Secondary structure of AAV TuD cassette specific for mir-26a. (A) Forward and Reverse strands of template DNA that were annealed to make the 26a-TuD expression cassette. The yellow color represents the mir-26a binding sequences (MBS) and the blue represents the stem II. Below these are the sequences that are part of the entry vector and flanked the forward template. (B) The expressed RNA including the flanking sequences (stem I) fold into the secondary structure determined by the CentroidFold software. The MBS is indicated. Suppl. Figure 9: Schedule for AntagomiR, BrdU and TuD injection and tissue collection. Time course of AntagomiR & BrdU (A) or TuD & CTX (B) injection and tissue harvesting is shown. Suppl. Figure 10: AAV expressing NC-TuD or 26a-TuD infect nearly all cells including Pax7-positive cells in adult muscle, as detected by expression of GFP. GFP immunostaining was carried out in the muscle sections expressing (A) NC-TuD (upper row) and 26a-TuD (lower row). indicates an uninfected muscle fiber. (B) Co-staining of GFP and Pax7 in 26a-TuD injected muscle. Yellow arrows: co-stained satellite cells. (C) FISH for mir-26a and 3

4 immunostaining for GFP in day 7 post CTX. MiR-26a remained up-regulated in the cells that were not infected with AAV 26a-TuD expressing GFP while mir-26a was decreased in cells where GFP was expressed. Suppl. Figure 11: 26a-TuD-injected muscle samples shows smaller myofibers. Additional images from the muscles in Fig. 6I. Desmin (green) and laminin (red) staining are performed on regenerating fibers on day 7 (A-C) and day 14 (D-F) post CTX injection. NC-TuD or 26a-TuD and cardiotoxin were injected as in Fig. 6H. Suppl. Figure 12: MiR-26a target sites on human Smad1 and Smad4 3 UTR. The RNA miranda algorithm predicted mir-26a (top) matching target sites (bottom) on Smad1 3 UTR and Smad4 3 UTR. 4

5 Dey _Suppl Table1 Suppl Table 1: Effect of overexpression and inhibition of mir-26a on myoblast differentiation. Transfectant Myog-positive Cells MHC-positive Cells Total counts (10 fields) % Control Total counts (10 fields) % Control Mean SD Mean SD GL mir-26a Anti-GL Anti-26a

6 Dey _Suppl Table 2 Table 2: List of qrt-pcr primers used in this study Primer Name Length Sequence U6sn 20 CTGCGCAAGGATGACACGCA mir-26a 22 TTCAAGTAATCCAGGATAGGCT mir CTGACCTATGAATTGACAGCC mir TGGAATGTAAGGAAGTGTGTGG Myogenin Forward 24 AGCGCAGGCTCAAGAAAGTGAATG Myogenin Reverse 24 CTGTAGGCGCTCAATGTACTGGAT MHC Forward 22 TCCAAACCGTCTCTGCACTGTT MHC Reverse 22 AGCGTACAAAGTGTGGGTGTGT Pax7 Forward 24 GAACCGTCTGGATGAGGGCTCAGA Pax7 Reverse 24 GCTCCTCCAGCTGCTCGGCTGTGA Id3 Forward 24 CTCTTAGCCTCTTGGACGACATGA Id3 Reverse 24 TGTAGTCTATGACACGCTGCAGGA Smad1 Forward 24 TGACCAGCTTGTTTTCATTCACAA Smad1 Reverse 24 GGCCCCTTTCTTCTTCTTCAGTTT Smad4Forward 24 AGCCATAGTGAAGGACTGTTGCAG Smad4 Reverse 24 TACTTCCAGTCCAGGTGGTAGTGC Ezh2Forward 24 CTCTGCCTCCTGAATGTACTCCAA Ezh2 Reverse 24 GGAAGGGATGTAGGAAGCAGTCAT GAPDH Forward 24 ATGACATCAAGAAGGTGGTGAAGC GAPDH Reverse 24 GAAGAGTGGGAGTTGCTGTTGAAG

7 Dey _Suppl Fig.1 A Control Day 7 Day 1 Day 10 Day 3 Day 14 Day 5 B mir-26a Pax7 DAPI/Overlay CTX Day 7

8 Dey _Suppl Fig GL2 mir-26a 50 % cells G1 S G2/M

9 Dey _Suppl Fig.3 A B Relative Myog and MHC mrna level GL2 mir-26a Relative Myog and MHC mrna level Anti-GL2 Anti-26a 0 Myog MHC 0 Myog MHC

10 Dey _Suppl Fig.4 A Smad1 Site 1: B Smad1 Site 2: C Smad1 Site 3: D Smad1 Site 4: E Smad4 Site 1: F Smad4 Site 2:

11 Dey _Suppl Fig.5 A B Smad1 GAPDH Relative Smad1 level (normalized to GAPDH)

Dey _Suppl Fig.6 A B 1.8 3.5 Relative Ezh2 level 1.6 1.4 1.2 1 0.8 0.6 0.

5 C 0 Control Ant26a D 0 NC-TuD 26a-TuD 2.5 2.

12 Dey _Suppl Fig.6 A B Relative Ezh2 level Relative Ezh2 level C 0 Control Ant26a D 0 NC-TuD 26a-TuD Relative Ezh2 level on Day NC-TuDCTX 26a-TuDCTX Relative Ezh2 level on Day NC-TuDCTX 26a-TuDCTX

13 DAPI Pax7 Ki67 Pax7/Ki67 Overlay/DIC Dey _Suppl Fig.7

14 A Dey _Suppl Fig.8 mir26atud Forward template: CATCAACAGCCTATCCTGGTCTCATTACTTGAACAAGTATTCTGGTCACAGAATACAACAGCCTATCCTGGTCTCATTACTTGAACAAG mir26atud Reverse template: TCATCTTGTTCAAGTAATGAGACCAGGATAGGCTGTTGTATTCTGTGACCAGAATACTTGTTCAAGTAATGAGACCAGGATAGGCTGTT For folding, forward template is flanked by GACGGCGCTAGGAT----ATGATCCTAGCGCCGTC B Stem I MiR-26a binding sites MiR-26a binding sites Stem II

15 Dey _Suppl Fig. 9 A Injection Age (Days) AntagomiR AntagomiR AntagomiR BrdU 2 hr Harvesting tissues B Injection AAV CTX 10 days Tissue harvesting days

16 Dey _Suppl Fig. 10 A DAPI DIC GFP 26a-TuD NC-TuD B DAPI DIC Pax7 GFP 26a-TuD C mir-26a GFP DAPI/Overlay

17 Dey _Suppl Fig.11 A B C NC-TuD CTX Day 7 26a-TuD CTX Day 7 26a-TuD CTX Day 7 D E F NC-TuD CTX Day 14 26a -TuD CTX Day 14 26a -TuD CTX Day 14

18 Dey _Suppl Fig.12 3' ucggauaggaccua-augaacuu 5' hsa-mir-26a : 31:5' auugagccuugcauguacuugaa 3' SMAD1 3' ucggauaggaccua--augaacuu 5' hsa-mir-26a 87:5' aaaggagccuugauaauacuugac 3' SMAD1 3' ucggauaggaccuaaugaacuu 5' hsa-mir-26a :: 1022:5' uggauauuuuug--uacuugau 3' SMAD4

SUPPLEMENTAL FIGURE LEGENDS

SUPPLEMENTAL FIGURE LEGENDS Suppl. Fig. 1. Notch and myostatin expression is unaffected in the absence of p65 during postnatal development. A & B. Myostatin and Notch-1 expression levels were determined