Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 1. Dusp13 is a novel target regulated by MyoD. (A) Clustering heatmap showing differentially expressed genes in RNA-seq data from Pax7- YFP (Quiescent MuSCs; QSCs), MyoD-low (MyoD-tdTomato low), and MyoD-high (MyoD-tdTomato high) (n = 3 per group). (B) Clustering heatmap of differentially expressed genes in RNA-seq between wild-type (WT) growth medium (GM) (WT_GM), MyoD knock-out GM (MKO_GM), WT differentiation medium (WT_DM), and MKO_DM (n = 3 per group). (C) Venn diagrams of indicated clusters from (A, B) and ChIP atlas top 100 genes potentially regulated by MyoD (Left). Venn diagrams of overlapped 35 genes (left) and 80 genes upregulated by MyoD overexpression in MEFs (Right). (D) List of 6 genes from (C). (E, F) Gene expression of MyoD (E) or Dusp13 (F) analyzed by RNA-seq in the Pax7-YFP, MyoD-low, and MyoD-high MuSCs (n = 3 per group). (G, H) Gene expression of MyoD (G) or Dusp13 (H) analyzed by RNA-seq in the WT_GM, WT_DM, MKO_GM, and MKO_DM groups (n = 3 per group). (I) E-box-like sequences in the promoter region of Dusp13 in mammalian species. (J) Luciferase reporter constructs of Dusp13 upstream region (−150 to +50) containing an E-box-like sequence (Top). Relative Dusp13-luciferase reporter activities in HEK293T cells overexpressing MyoD (Bottom) (n = 3 per group). All data are presented as mean ± SEM P values calculated using edge R (E, F) (FDR-corrected), DEseq2 (G, H) (FDR- corrected), or Tukey’s test (J); **P < .01, ***P < .001, n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: RNA Sequencing, Knock-Out, Over Expression, Gene Expression, Luciferase, Construct, Sequencing

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 2. Absence of skeletal muscle phenotype in Dusp13 knock-out (13 KO) mice during development and regeneration. (A) Hematoxylin and eosin (H&E) staining of Soleus (SOL) and Plantaris (PLA) muscle cross-sections of male Dusp13 knock-out (13 KO) and WT mice at 10 weeks. Scale bar: 100 μm. (B, C) Cross-sectional area (CSA) of SOL (B) or PLA (C) (n = 3 per group). (D) Immunofluorescence of PAX7 and LAMININ in intact TA muscles. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (E) Average number of PAX7 (+) nuclei in DAPI (+) in (D). (F) H&E staining of tibialis anterior (TA) muscle cross-sections of male 13 KO and WT mice at 12 weeks, 7 days post cardiotoxin (CTX) injury. Scale bar: 100 µm. (G) CSA of damaged TA muscles (n = 3 per group). (H) Immunofluorescence of PAX7 with LAMININ on sections of TA muscles from male WT and Dusp13 KO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (I) Average number of PAX7 (+) nuclei in (H) are quantified (n = 3–4 independent experiments). (J) Immunofluorescence of MYOGENIN with LAMININ on sections of TA muscle from male WT and 13 KO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (K) Average number of MYOGENIN (+) nuclei in (J) are quantified (n = 3–4 independent experiments). All data are presented as mean ± SEM P values calculated using Student’s t-test; n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Knock-Out, Staining, Immunofluorescence, Muscles

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 3. Dusp27 KO mice exhibit comparable muscle development and regeneration to WT mice. (A) qPCR analysis of Dusp27 mRNA levels in gastrocnemius muscles of male WT and 13 KO mice at 12 weeks (n = 3 per group). (B) Gene expression of Dusp27 in RNA-seq data from Pax7-YFP, MyoD-low, and MyoD-high groups (n = 3 per group). (C) E-box-like sequences in the promoter region of Dusp27 in mammalian species. (D) Luciferase reporter constructs of Dusp27 upstream region (−1000 to +200) containing an E-box-like sequence (Top). Relative Dusp27-luciferase reporter activities in HEK293T cells overexpressing MyoD (Bottom) (n = 3 per group). (E, G) H&E staining of SOL and PLA muscle cross-sections. Scale bar: 100 μm. (F, H) CSA of SOL (F) or PLA (H) muscles of male Dusp27 KO (27 KO) and WT (n = 3–6 independent experiments) mice at 12 weeks. (I) Immunofluorescence of PAX7 and LAMININ in intact TA muscles. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (J) Average number of PAX7 (+) nuclei in DAPI (+) in (I). (K) H&E staining of TA muscle cross-sections of male 27 KO and WT mice at 12 weeks (n = 5–6

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Muscles, Gene Expression, RNA Sequencing, Luciferase, Construct, Sequencing, Staining, Immunofluorescence

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 4. Loss of both Dusp13 and Dusp27 impairs muscle regeneration after acute muscle injury. (A) H&E staining of tibialis anterior (TA) muscle cross- sections of male Dusp13 and Dusp27 double knock-out (DKO) (n = 3) and WT (n = 3) mice at 12-15 weeks of age, 3, 7, and 28 days post-cardiotoxin (CTX) injury. Scale bar: 100 µm. (B–D) Cross-sectional area (CSA) of damaged TA muscles at indicated time points. (E, F) Immunofluorescence of PAX7 (E) or MYOGENIN (F) with LAMININ on sections of TA muscles from male WT and DKO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (G) Iimmunofluorescence of KI67 and MYOGENIN with LAMININ on sections of TA muscles from male WT and DKO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. Arrowheads indicate KI67+/MYOGENIN + cells. Arrows indicate KI67-/MYOGENIN + cells. (H, I) Average number of PAX7 (+) or MYOGENIN (+) nuclei in (E) and (F) are quantified (n = 5-8 independent experiments). (J) Frequency of Ki67 (+) nuclei in MYOGENIN (+) in (G) are quantified (n = 5 per group) (K) Immunofluorescence of PAX7 and MYOD on cultured MuSCs from female WT and DKO mice. Nuclei are stained with DAPI. Scale bar: 100 µm. (L, M) Quantification of the proportion (L) and numbers (M) of MuSCs undergoing self-renewal (PAX7 (+), MYOD (−)), activation (PAX7 (+), MYOD (+)), and differentiation [PAX7 (+), MYOD (−)] in (K).

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Staining, Knock-Out, Muscles, Immunofluorescence, Cell Culture, Activation Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 6. Overexpression of Dusp13 induces precocious muscle differentiation independent of phosphatase activity. (A) Schematic diagram illustrating adenovirus infection into MuSCs from 15 to 16-week-old male Pax7-YFP mice on day 4 of growth medium (GM) culture. (B) Number of cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP). (C) Immunofluorescence staining of PAX7 and MYOGENIN in cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP) on GM day 4. Nuclei are stained with DAPI. Arrows indicate PAX7 (+)/MYOGENIN (+) cells. (n = 6 per group). Scale bar: 100 µm (upper); 25 µm (bottom). (D) The proportion of each population in (C). (E) Schematic diagram illustrating adenovirus infection into MuSCs from 15 to 16-week-old male Pax7-YFP mice on GM day 9. (F) Immunofluorescence staining of MYOGENIN in cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP) on GM day 9. Nuclei are stained with DAPI. Scale bar: 100 µm (G, H) Myotube width (G) and Fusion index (numbers of myonuclei per myotube) (H) in (F) are quantified (50 myotubes analyzed per each experiment) All data are presented as mean ± SEM. P values calculated using Student’s t-test; *P < .05, **P < .01, ***P < .001, n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Over Expression, Activity Assay, Infection, Cell Culture, Transduction, Immunofluorescence, Staining