Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 1 MuRF1 and MuRF2 are highly induced during skeletal muscle regeneration. (a) MuRF1, MuRF2 and Mafbx/Atrogin‐1 immunoblots 3 and 10 days after TA CTX injury. Sarcomeric actin is a loading control. (b‐g) Immunolocalization of MuRF1 and (j‐o) MuRF2, 3 and 10 days after CTX injury in TA muscle. In h‐i and p‐q, primary antibodies were not used in the assay. Immunodetection of laminin (green) was used to outline muscle fibers. (h‐i and p‐q) No primary antibodies were added to the assay. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 50µm

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Western Blot, Control, Immunodetection, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 2 Combined deletion of MuRF1 and MuRF2 causes deficient skeletal muscle regeneration. TA muscles from WT (a and e), from MuRF1 KOs (b and f), MuRF2 KOs (c and g), or from MuRF1&2 dKOs (d and h) were CTX injured and histologically analyzed 3 or 10 days later, respectively. We also histologically analyzed WT and MuRF1&2 dKO mice at 28 days (i and j respectively) after CTX. TA from MuRF1&2 dKO mice presented indications of defective regeneration in all time points analyzed (d, h and j, see text for detailed description). Bar, 20µm. (k) Average density of necrotic fibers in WT and MuRF1&2 dKO 24h after CTX injury; error bars indicate standard deviations (N=3)

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Muscles

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig.3 Simultaneous deletion of MuRF1 and MuRF2 decreases the number of cells positive to Pax7 and Myod. TA muscles from WT, or MuRF1&2 dKO mice were investigated by immunofluorescence 3 (a‐d and j‐m) or 10 days (e‐h and n‐q) after CTX injections. Immunolocalization and percentage of positive eMHC positive myofibers was also determined (u). DAPI (blue), Pax7 and Myod (red), laminin (green) and eMHC (green). Images were obtained with a fluorescence microscope and a 40x air medium objective. Large bar, 50 µm, small bar 10 µm

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Muscles, Immunofluorescence, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 4 Simultaneous deletion of MuRF1 and MuRF2 causes decreased protein expression of myogenic factors and increased apoptosis. (a) Western blots for Myogenin, Myf‐5, FHL2, MARP2 and GAPDH in TA of MuRF1&2 dKO animals 3 and 10 days after CTX injury and silver staining showing even protein loading. Fluorescence images showing apoptotic nuclei by TUNEL assay in WT (b‐d and h‐j) and MuRF1&2 dKO (e‐g and k‐m) muscles 3 (b‐g) and 10 (h‐ m) days after CTX injury. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 30 µm. (n) Percentage of apoptotic nuclei in WT and MuRF1&2 dKO muscles 3 and 10 days after CTX injury

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Expressing, Western Blot, Silver Staining, Fluorescence, TUNEL Assay, Muscles, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 5 Simultaneous deletion of MuRF1 and MuRF2 time‐shifts the localization/expression of β‐catenin during regeneration. TA muscles from WT (a‐e and k‐o), or MuRF1&2 dKO mice (f‐j and p‐t) were studied by immunofluorescence 3 (a‐j) or 10 days (k‐t) after CTX injections. DAPI (blue), β‐catenin (red), MyoD (green) and laminin (white). Arrows indicate nuclei simultaneously positive to MyoD, B‐catenin and Dapi. Arrow heads indicate non‐nuclear β‐catenin labeling. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 20 µm. (u) Number of β‐catenin positive cells/mm 2 in WT and MuRF1&2 dKO TA muscles. ***p<0.001 vs WT

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Expressing, Muscles, Immunofluorescence, Labeling, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 6 siRNA knock down of MuRF1 and MuRF2 reduce myogenesis. (a‐d) Phase contrast micrographs of primary myoblast culture 2 days after induction of differentiation. Images were taken with a 4x objective. Cells were siRNA knocked down for MuRF1 (b), MuRF2 (c) and MuRF1 and MuRF2 combined (d). (a) Arrowheads indicate the normal differentiation pattern generating myotubes. (b‐d) Arrows indicate a severe differentiation deficit. (e) Fusion index of siRNA knocked down cells. (f) Myotube area of siRNA knocked down cells. (g) Number of myotube by field of siRNA knocked down cells. The fusion index was calculated as the ratio of the nuclei number in myotubes with two or more nuclei versus the total number of nuclei. Ten representative images per sample were scored for myotube number and area occupied by myotubes relative to the total area ( a p<0.05 vs Control). Fluorescence images showing reduced expression of eMHC after siRNA knock down for MuRF1 (k‐m) MuRF2 (n‐p) and MuRF1 and MuRF2 combined (q‐s) compared to control (h‐j). Images were obtained with a fluorescence microscope. Bars, 20 µm

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Knockdown, Control, Fluorescence, Expressing, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 7 siRNA knock down of MuRF1 and MuRF2 reduce MyoD positive cell number. Cells were siRNA knocked down for MuRF1 (e‐h), MuRF2 (i‐l) and MuRF1 and MuRF2 combined (m‐p). Immunoflurescence for Pax7 (red) and MyoD (green) and dapi staining was performed. (q) Percentage of positive nuclei for MyoD. (r) Percentage of positive nuclei for Pax7. (s) Percentage of positive nuclei for Pax7 and MyoD. Ten representative images per group were scored for percentage of positive nuclei (*p<0.05 vs Control). Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 30 µm

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Knockdown, Staining, Control, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 8 siRNA knock down of MuRF1 and MuRF2 induce accumulation of BAF57 in the nucleus of primary myoblasts culture 2 days after induction of differentiation. (a‐j) Immunolocalization of BAF57 in cells siRNA knocked down for MuRF1 (c and d), MuRF2 (e and f) and MuRF1 and MuRF2 combined (g and h). (i) Representative immunoblots for BAF57 and GAPDH in cytoplasmic fraction from cells siRNA knocked down for MuRF1 (MuRF1 RNAi), MuRF2 (MuRF2 RNAi) and MuRF1 and MuRF2 combined (MuRF1/2 RNAi). (j) Representative immunoblots for BAF57 and GAPDH in nuclear fraction from cells siRNA knocked down for MuRF1 (MuRF1 RNAi), MuRF2 (MuRF2 RNAi) and MuRF1 and MuRF2 combined (MuRF1/2 RNAi). Bar, 40 µm

Article Snippet: The primary antibodies used for western blot were: (1) MuRF1 and (2) MuRF2 [27, 32]; (2) Atrogin/Mafbx‐1 (Cat#AP2041, ECM Biosciences); (3) Myf‐5 (Cat#sc‐302, Santa Cruz); (4) Myogenin (Cat#ab1835, Abcam); (5) sarcomeric actin (Cat#M0847, Dako); (6) GAPDH (Cat# 2118, CellSignaling), (7) FHL2 (Cat# A300‐332A, Bethyl Laboratories); (8) MARP2; (9) BAF57 (Cat#Abd52, Millipore).

Techniques: Knockdown, Western Blot