1. Muscle Satellite Cells Are Primed for Myogenesis but Maintain Quiescence with Sequestration of Myf5 mRNA Targeted by microRNA-31 in mRNP Granules 
Colin G. Crist, Didier Montarras, Margaret Buckingham 
Cell Stem Cell 
Volume 11, Issue 1, Pages (July 2012)
DOI: /j.stem
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Cell Stem Cell 2012 11, 118-126DOI: (10.1016/j.stem.2012.03.011)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Myf5 and miR-31 Expression in Quiescent and Activated Satellite Cells
(A) Immunostaining with antibodies against β-galactosidase (β-gal; red) on a freshly isolated (0 hr) EDL muscle fiber from a Myf5nlacZ/+ mouse. Right panel shows merged image with DAPI.
(B) Immunostaining with Pax7 (green, top) and Myf5 (red, middle) antibodies on a freshly isolated (0 hr) wild-type EDL fiber.
(C and D) Immunostaining on EDL fibers, after 24 hr of culture, with Pax7 (green, top left), Myf5 (red, middle), and MyoD (green, top right) antibodies. Merged images with DAPI are shown in the bottom panels of (B), (C), and (D).
(E) Western blot analysis of Myf5 protein levels (normalized to β-tubulin, β-tub) (top) with quantitative PCR analysis of Myf5 transcripts (normalized to Actb transcripts) (middle) and miR-31 levels (normalized to U6 snRNA) (bottom) in satellite cells directly isolated (day 0) from adult Pax3GFP/+ (wt), adult Pax3GFP/+; mdx:mdx (mdx), or postnatal day 7 (P7) Pax3GFP/+ mice. Error bars represent standard error of the mean (SEM) of three replicate experiments.
(F–I) Sections of adult Tibialis anterior (TA) muscle showing miR-31 (F; purple) detected by ISH in a satellite cell (arrows) marked by DAPI or by ISH with a scrambled oligonucleotide probe (G; scramble, purple). Scale bars represent 20 μm. Combined detection of proteins by immunostaining of TA sections with antibodies to Pax7 and laminin (left), with miRNA ISH (middle) for miR-31 (H; purple, arrowheads), or a scrambled probe (I). Merged images are shown in the right panels. Satellite cells, marked by a Pax7-positive nucleus (green), are indicated by arrows. Asterisks (∗) in (F) and (H) mark adjacent fibers, negative for miR-31.
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4. Figure 2
mRNP Granules Contain Myf5 Transcripts and miR-31 in the Quiescent Satellite Cell
(A–C) Immunostaining with Pax7, Tia1, p54/RCK, and HuR antibodies on EDL fibers shows Tia1 (red) (A) and p54/RCK (red) (B) localization to cytoplasmic granules in a quiescent satellite cell marked by Pax7 (green). HuR (green) remains in the nucleus of a quiescent satellite cell identified by p54/RCK foci (C).
(D–F) After 24 hr of culture, Tia1 (red) (D) and p54/RCK (red) (E) show diffuse cytoplasmic localization in activated satellite cells marked by MyoD-positive (green) nuclei. p54/RCK and HuR show diffuse cytoplasmic (red) and nuclear (green) staining, respectively (F). Insets in (A)–(F) show enlargements of the satellite cells without DAPI staining of the nucleus.
(G) Fluorescent in situ hybridization (FISH) for miR-31 (top), Myf5 transcripts (middle), and a scrambled-miR probe (scramble, bottom) on quiescent satellite cells freshly isolated from Pax3GFP/+ adult muscle.
(H) Immunofluorescent labeling on sections of adult TA muscle; p54/RCK (red) shows the presence of cytoplasmic mRNP granules in a satellite cell, outlined by characteristic dystrophin labeling (green) of the contour of the muscle fiber (top). The same satellite cell is shown enlarged, labeled for p54/RCK and DAPI (middle), followed by ISH for miR-31 (purple, bottom). Arrows denote the nucleus.
(I) Cytoplasmic fractions from freshly isolated in vivo quiescent satellite cells from adult Pax3GFP/+ muscle (left) and in vivo activated satellite cells from Pax3GFP/+; mdx:mdx muscle (right) were analyzed by RT-PCR for Pax3, Myf5, and Actb transcripts (top) or by qRT-PCR for miR-31, normalized to U6 snRNA (bottom). Error bars represent SEM of three replicate experiments. T, total cell fraction; S, soluble cytoplasmic fraction; P, pelletable cytoplasmic fraction containing mRNP granules. Increased concentration of mRNP granules in the pelletable compared to soluble cytoplasmic fraction from quiescent satellite cells is shown by western blotting with antibodies against p54/RCK (middle).
(J) Quantitation of coexpression of Pax7 and Myf5, monitored by immunostaining, on satellite cells of freshly isolated EDL fibers cultured in the presence of 10 μg/ml α-amanitin (green) or 100 μg/ml cycloheximide (CHX, red). Error bars represent SEM of three independent single fiber cultures. See also Table S1 and Figure S1.
(K) Quantitation of the number of mRNP granules upon activation of satellite cells (marked by Pax7) on cultured EDL fibers over time, visualized by immunostaining with antibodies against p54/RCK. Error bars represent SEM with n > 50 at each time point.
(L and M) Immunostaining with Pax7 (green), p54/RCK (red), and Myf5 (red) antibodies on satellite cells after 1 hr of EDL fiber culture, in the absence (control) or presence of 1 mM sodium arsenite.
(N and O) Quantification of the number of granules per Pax7-positive satellite cell on isolated EDL fibers (N) and percentage of Pax7-positive satellite cells that show immunofluorescence for Myf5 in these satellite cells (O). 0, freshly isolated fibers; C, control fibers after 1 hr culture; Ars, fibers cultured for 1 hr in the presence of 1 mM sodium arsenite. Error bars represent SEM.
(P) Immunostaining with Pax7, p54/RCK, FMRP, and phosphospecific FMRP (P-FMRP) antibodies on freshly isolated (0 hr) EDL fibers. Arrows show FMRP localization to p54/RCK (red) containing granules and P-FMRP (red) localization to granules in quiescent satellite cells where the nucleus is marked by Pax7 (green).
(Q) Immunostaining with P-FMRP (red) and Pax7 (green) antibodies after 3 hr of EDL fiber culture in the absence (control) or presence of 10 nM Okadaic acid (OA). Arrows point to P-FMRP in granules.
(R and S) Percentage of Pax7-positive satellite cells that show immunofluorescence for P-FMRP (R) and Myf5 (S) in satellite cells on 0, freshly isolated fibers, or 3, fibers after 3 hr culture in the absence or presence of 1 or 10 nM OA, as indicated. Error bars represent SEM.
See also Figure S2.
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5. Figure 3
Differentiation of Activated Satellite Cells Is Modulated by miR-31 Regulation of Myf5 Protein Levels
(A and B) Detection of Myf5 (green) and Myogenin (MyoG) (red) by immunocytochemistry with the corresponding antibodies after satellite cells were transfected with control (A) or pre-miR-31 (B) precursor oligonucleotides and cultured for 3 days.
(C and D) After 4 days in culture, immunocytochemistry with antibodies against MyoG (green) and TroponinT (TropT, red) on control or pre-miR-31-transfected satellite cells.
(E) The percentage of satellite cells expressing Myf5, Myogenin, and TroponinT, monitored by immunocytochemistry, after 3 and 4 days in culture, of control-transfected (C) and pre-miR-31-transfected (31) satellite cells isolated from adult Pax3GFP/+ mice. Error bars represent SEM of three independent cultures with p < 0.001 using paired Student's t test. See also Figure S3.
(F–I) Detection of MyoG (green) with MyoD (red) (F and G) or TropT (red) (H and I) by immunocytochemistry with the corresponding antibodies after satellite cells isolated from adult Myf5−/− mice were transfected with control (F and H) or pre-miR-31 (G and I) precursor oligonucleotides and cultured for 3 (F and G) or 4 (H and I) days.
(J) Percentage of satellite cells, isolated from adult Myf5−/− mice, expressing MyoD, Myogenin, and TroponinT, monitored by immunocytochemistry, after 3 and 4 days in culture, after transfection with pre-miR-31 (31) and control (C) oligonucleotides. Error bars represent SEM of three independent cultures.
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6. Figure 4
Satellite Cell Activity and Skeletal Muscle Regeneration Is Modulated by miR-31 Regulation of Myf5
(A–I) Uninjured TA muscle after antagomiR-31 treatment.
(A) Immunostaining of sections with antibodies against Pax7 (top, green) and laminin (middle, red), with DAPI staining of nuclei, in mice treated with antagomiRs against miR-31 (antagomiR-31) or a control antagomiR. Merged images are shown in the bottom panels. Arrowheads point to satellite cells.
(B) Immunostaining of sections with antibodies against Pax7 (green) and Myf5 (red) in mice treated with antagomiR-31 or a control antagomiR. Merged images, with DAPI staining, are shown in the bottom panels. Colocalization of Myf5 with Pax7 (yellow) in a satellite cell of antagomiR-31-treated mice is indicated (arrowheads).
(C) Immunostaining of sections with antibodies against Pax7 (green) and p54/RCK (red) of antagomiR-31, compared to control antagomiR-treated animals. Insets show enlargement of satellite cells with DAPI staining of the nucleus.
(D) Immunostaining of sections with antibodies against MyoD (red) and Ki67 (green), with DAPI staining of the nuclei, of antagomiR-31, compared to control antagomiR-treated mice.
(E and F) Quantification of the number of satellite cells expressing Pax7 (E) and Myf5 (F), per 100 fibers, on such transverse sections of TA muscle from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent SEM.
(G) Percentage of Pax7-positive satellite cells with cytoplasmic mRNP granules labeled by p54/RCK on transverse sections of TA muscles from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent SEM.
(H) Quantification of the number of myonuclei per 100 fibers on sections of TA muscle from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent SEM.
(I) The mean TA muscle fiber cross-section area (CSA) from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent 95% confidence interval surrounding the mean with p < using paired Student's t test.
(J–P) Injured TA muscle after antagomiR-31 treatment.
(J) Immunostaining of sections with antibodies against Myf5 (red) and myosin heavy chain (MHC, green) 10 days after cardiotoxin injury in untreated (left) or antagomiR-31-treated (right) animals.
(K) Immunostaining of sections with antibodies against embryonic myosin heavy chain (embMHC, green) and laminin (red) 10 days after cardiotoxin injury in control antagomiR (left) or antagomiR-31-treated (right) animals. Scale bars in (J) and (K) represent 20 μm.
(L) The mean CSA of embMHC-positive TA fibers, 10 days after cardiotoxin injury, from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent 95% confidence interval surrounding the mean with p < using paired Student's t test.
(M) Immunostaining of sections with antibodies against Pax7 (red) and laminin (Lam, green) 21 days after cardiotoxin injury. The position of satellite cells underneath the basal lamina is marked with white arrowheads. Scale bars represent 20 μm.
(N) The mean CSA of regenerating TA fibers (central nuclei), 21 days after cardiotoxin injury, from mice treated with control antagomiR (C) or antagomiR-31 (α31). Error bars represent 95% confidence interval surrounding the mean with p < using paired Student's t test.
(O) Immunostaining of sections, focusing on satellite cells, with antibodies against p54/RCK (red) and Pax7 (green), 21 days after cardiotoxin injury (Ctxn) and in a contralateral uninjured muscle. Nuclei in sections (J), (K), (M), and (O) are stained with DAPI (blue).
(P) Percentage of Pax7-positive satellite cells containing cytoplasmic mRNP granules as indicated by immunostaining with antibodies against p54/RCK, 21 days after cardiotoxin injury, in untreated (C) and antagomiR-31-treated (α31) animals. Error bars represent SEM.
See also Figures S3 and S4.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2012 Elsevier Inc. Terms and Conditions