Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Gata4, Mef2c, and Tbx5 expression in the beating and non-beating groups. ( a ) Left three panels, gene expression levels in the beating and non-beating group. Right panel, gene expression levels in the beating and non-beating group relative to that of β-actin. ( b ) Representative immunofluorescence images of a cluster of round cells expressing Gata4 and Mef2c on day 14. ( c ) Phase-contrast and immunofluorescence images of beating cells expressing cardiac troponin T (cTnT) and Mef2c on day 28. The arrowhead indicates a beating cell. Scale bar, 100 µm. DAPI, 4′,6-diamidino-2-phenylindole, ** p < 0.01; **** p < 0.0001 by t -test or ANOVA and Tukey’s post hoc test. ( d ) The frequency of round cells expressing Gata4 and Mef2c (10 round cells were examined per experiment, n = 13). ( e ) The frequency of beating cells expressing Mef2c and cTnT (15 beating cells were analysed per experiment, n = 4).

Article Snippet: For silencing of mouse Mef2c, LentiORF lentiviral particles of mouse Mef2c-specific short hairpin RNA (TL511428V, Origene) or scrambled control were used.

Techniques: Expressing, Gene Expression, Immunofluorescence

Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Overexpression of SVF with Mef2c augments the number of round cells and their transdifferentiation into CMs. ( a – d ) Phase-contrast images of SVF on day 28 after transfection on day 5 ( a , control; b , Gata4; c , Mef2c; d , Tbx5). Arrowheads indicate round cells; lv, lentivirus vector. ( e ) mRNA level of Mef2c, determined by qRT-PCR, on day 28 in SVFs transfected with the control vector and Mef2c-lv (left), or scrambled shRNA and shMef2c-lv (right), on day 5, respectively. shRNA, short hairpin RNA; shMef2c, shRNA targeting Mef2c. ( f ) Number of round cells on day 14 after introducing SVF with none (none) or the control, Gata4, Mef2c, shMef2c, or Tbx5 lentivirus vector. ( g ) mRNA level of cardiac troponin T by qRT-PCR on day 28 in SVF transfected with the control, Gata4, Mef2c, shMef2c, or Tbx5. ( h ) Relationship between the mRNA levels of Mef2c and cardiac troponin T in the SVF transduced with Mef2c. R, Pearson’s product-moment correlation coefficient. The linear interpolation formula is displayed. Scale bar = 100 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001 by ANOVA and Tukey’s post hoc test.

Article Snippet: For silencing of mouse Mef2c, LentiORF lentiviral particles of mouse Mef2c-specific short hairpin RNA (TL511428V, Origene) or scrambled control were used.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, shRNA, Transduction

Journal: Scientific Reports

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

doi: 10.1038/s41598-017-19114-y

Figure Lengend Snippet: POU5F1 knockdown facilitates synMYOD1-induced myogenic gene activation. ( a ) Immunostaining analysis for POU5F1 in the siPOU5F1 and synMYOD1 (siPOU5F1/synMYOD1)-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( b ) Immunostaining analysis for NANOG in the siPOU5F1/synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( c ) Immunoblotting analysis for POU5F1 in the siPOU5F1/synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and siPOU5F1/synMYOD1 transfection (mean ± SEM from three independent biological replicates). * P < 0.01, t-test. ( d ) ChIP analysis showing POU5F1 enrichment at the promoter regions (Pro-1 and Pro-2) of POU5F1 in hESCs, siControl/synMYOD1-treated cells, and siPOU5F1/synMYOD1-treated cells. The promoter regions of MEF2C and MYOG were used as negative controls. The error bars indicate the SEM from three independent biological replicates. * P < 0.05, t-test. ( e ) ChIP analysis showing MYOD1 enrichment at the promoter regions of MEF2C and MYOG in hESCs, siControl/synMYOD1-treated cells, and siPOU5F1/synMYOD1-treated cells. mRNA encoding HA-tagged MYOD1 and anti-HA antibody was used in this experiment as the anti-MYOD1 antibody suitable for ChIP was not available. The promoter regions of POU5F1 were used as negative controls. The error bars indicate the SEM from two independent biological replicates. * P < 0.05, t-test. NS: not significant. ( f ) qPT-PCR analysis for the expression of myogenic markers in the siControl (siC), siPOU5F1 (siP), siC/synMYOD1, and siP/synMYOD1-treated cells. The expression levels were normalized to GAPDH . The error bars indicate the SEM from two independent biological replicates. * P < 0.01, t-test. Uncropped images of the blots for Fig. 2c are shown in Supplementary Figure .

Article Snippet: The following antibodies were used: POU5F1 (Abcam #ab19857 and Santa Cruz #sc-5279), NANOG (Abcam #ab21624), MyHC (R&D #MAB4470), SOX2 (Millipore #AB5603), MYC (Abcam #ab32072), T (R&D #AF2085), PAX7 (Invitrogen #PA1-117), MYOG (Abcam #ab124800), MEF2C (CST #5030, DSHB #1D4), SIX1 (CST #12891), MYH2 (Sigma #M1570), MYH3 (DSHB #F1.652), MYH8 (DSHB #N3.36), TTN (DSHB #9D10), ACTN2 (Sigma #A7811), DES (Abcam #ab32362), TNNT2 (Santa Cruz #sc-20025), MYOD1 (BD #554130 and Abcam #ab21624), HA (Abcam #ab18181), and H3 (Abcam #ab1791), and β-actin (Cell Signaling #4970S).

Techniques: Knockdown, Activation Assay, Immunostaining, Transfection, Staining, Western Blot, Control, Expressing

Journal: Scientific Reports

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

doi: 10.1038/s41598-017-19114-y

Figure Lengend Snippet: siPOU5F1/synMYOD1 treatment induces efficient myogenic conversion of hPSCs. ( a ) Schematic of the myogenic differentiation protocol. hPSCs were transfected with siPOU5F1 together with synMYOD1 at the indicated time points. The mixture of siPOU5F1/synMYOD1 was transfected on day 0. synMYOD1 was transfected on days 1 and 2. The cells were cultured in αMEM + 5% KSR. ( b ) Morphological changes in the transfected cells. Scale bar, 50 μm. ( c ) Immunostaining analysis for MyHC in the hPSCs (SEES3 ESCs and 409B2 iPSCs) after the treatment with siControl/synMYOD1 or siPOU5F1/synMYOD1. Nuclei were stained with DAPI. The representative images are shown. The average percentages of MyHC-stained cells are obtained from three independent biological replicates. Scale bar: 200 μm. ( d ) Immunostaining analysis for MyHC in the hPSCs (H9 ESCs, 201B7 iPSCs, and TkDA3-4 iPSCs) after treatment with siControl/synMYOD1 or siPOU5F1/synMYOD1. Nuclei were stained with DAPI. The representative images are shown. The average percentages of MyHC-stained cells are obtained from three independent biological replicates. Scale bar: 200 μm. ( e ) Immunostaining analysis for MYOG, MEF2C, SIX1, MYH3, MYH8, titin (TTN), troponin T (TNN2), actinin alpha 2 (ACTN2), and desmin (DES) in the siPOU5F1/synMYOD1-treated cells. Scale bar: 50 μm. ( f ) Higher magnification of ACTN2 staining. Scale bar: 20 μm. ( g ) 409B2-iPSCs expressing Emerald GFP were differentiated into myogenic cells and co-cultured with mouse C2C12 myotubes, nuclei of which were labeled with red fluorescence. Next day after co-culturing, cell fusions were detected. Scale bar: 50 μm.

Article Snippet: The following antibodies were used: POU5F1 (Abcam #ab19857 and Santa Cruz #sc-5279), NANOG (Abcam #ab21624), MyHC (R&D #MAB4470), SOX2 (Millipore #AB5603), MYC (Abcam #ab32072), T (R&D #AF2085), PAX7 (Invitrogen #PA1-117), MYOG (Abcam #ab124800), MEF2C (CST #5030, DSHB #1D4), SIX1 (CST #12891), MYH2 (Sigma #M1570), MYH3 (DSHB #F1.652), MYH8 (DSHB #N3.36), TTN (DSHB #9D10), ACTN2 (Sigma #A7811), DES (Abcam #ab32362), TNNT2 (Santa Cruz #sc-20025), MYOD1 (BD #554130 and Abcam #ab21624), HA (Abcam #ab18181), and H3 (Abcam #ab1791), and β-actin (Cell Signaling #4970S).

Techniques: Transfection, Cell Culture, Immunostaining, Staining, Expressing, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

doi: 10.1038/s41598-017-19114-y

Figure Lengend Snippet: POU5F1 binding profiles around the myogenic gene loci and the effect of POU5F1 knockdown on the expression of the genes. ( a ) ChIP-sequencing tracks of POU5F1 for the loci of myogenic genes: PAX3, PAX7, MEF2C, MYOD1, and MYOG. The POU5F1 locus is shown as a positive control for the POU5F1 binding sites (orange). The data was obtained from a previous study ( b ) Expression levels of myogenic genes in the siControl- and siPOU5F1-treated cells were analyzed by RNA-sequencing. The FPKM values are shown.

Article Snippet: The following antibodies were used: POU5F1 (Abcam #ab19857 and Santa Cruz #sc-5279), NANOG (Abcam #ab21624), MyHC (R&D #MAB4470), SOX2 (Millipore #AB5603), MYC (Abcam #ab32072), T (R&D #AF2085), PAX7 (Invitrogen #PA1-117), MYOG (Abcam #ab124800), MEF2C (CST #5030, DSHB #1D4), SIX1 (CST #12891), MYH2 (Sigma #M1570), MYH3 (DSHB #F1.652), MYH8 (DSHB #N3.36), TTN (DSHB #9D10), ACTN2 (Sigma #A7811), DES (Abcam #ab32362), TNNT2 (Santa Cruz #sc-20025), MYOD1 (BD #554130 and Abcam #ab21624), HA (Abcam #ab18181), and H3 (Abcam #ab1791), and β-actin (Cell Signaling #4970S).

Techniques: Binding Assay, Knockdown, Expressing, ChIP-sequencing, Positive Control, RNA Sequencing

Journal: Scientific Reports

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

doi: 10.1038/s41598-017-19114-y

Figure Lengend Snippet: A model of the mechanism for the effective myogenic differentiation of hPSCs with siPOU5F1 and synMYOD1. ( a ) In hPSCs, POU5F1 directly represses the expression of the genes related to extracellular matrix and early myogenic genes such as PAX3 and PAX7. Additionally, when synMYOD1 alone is introduced in hPSCs, POU5F1 inhibits the access of the translated MYOD1 protein to the target late myogenic genes such as MEF2C and MYOG, which results in the failure of myogenic differentiation. ( b ) Knockdown of POU5F1 with siPOU5F1 causes the activation of extracellular matrix genes and early myogenic genes. Consequently, when synMYOD1 is introduced, the translated MYOD1 protein can access to the promoters of the late myogenic genes to activate terminal myogenic gene activation. These mechanisms facilitate the rapid and highly efficient myogenic differentiation in hPSCs.

Article Snippet: The following antibodies were used: POU5F1 (Abcam #ab19857 and Santa Cruz #sc-5279), NANOG (Abcam #ab21624), MyHC (R&D #MAB4470), SOX2 (Millipore #AB5603), MYC (Abcam #ab32072), T (R&D #AF2085), PAX7 (Invitrogen #PA1-117), MYOG (Abcam #ab124800), MEF2C (CST #5030, DSHB #1D4), SIX1 (CST #12891), MYH2 (Sigma #M1570), MYH3 (DSHB #F1.652), MYH8 (DSHB #N3.36), TTN (DSHB #9D10), ACTN2 (Sigma #A7811), DES (Abcam #ab32362), TNNT2 (Santa Cruz #sc-20025), MYOD1 (BD #554130 and Abcam #ab21624), HA (Abcam #ab18181), and H3 (Abcam #ab1791), and β-actin (Cell Signaling #4970S).

Techniques: Expressing, Knockdown, Activation Assay

Journal: Nature Cardiovascular Research

Article Title: Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration

doi: 10.1038/s44161-025-00718-x

Figure Lengend Snippet: a , Representative images of immunofluorescence staining showing Mef2 + PCNA + double-positive proliferating cardiomyocytes in TU and WIK at 7 dpci. Framed areas highlight the wound border zone (red) (scale bar, 300 μm). b , Quantification of Mef2 + PCNA + double-positive cells showing differences in proliferating cardiomyocytes in the border zone at 7 dpci but not at 21 dpci. c , Positive correlation of percentage of proliferating cardiomyocytes in the border zone between 7 dpci and 21 dpci. d , No correlation between 7 dpci border zone proliferation and 90 dpci wound length. e , Venn diagram displaying complete lack of overlap between genes correlating to 7 dpci border zone proliferation and 90-dpci wound length. f , g , No correlation between border zone proliferation and OXPHOS ( f ) or Glycolysis ( g ) at 7 dpci. h , Quantification of Mef2 + PCNA + cells showing no difference in proliferating cardiomyocytes in the border zone of 7 dpci KCL adult treated with inhibitor PF-04859989 or rotenone compared to DMSO control. i , Temporal wound length reduction of all strains between 1, 7, 21 and 90 dpci. Arrow highlighting the strong decrease in wound length in WIK between 7 dcpi and 21 dpci. j , Percentage of hearts completely regenerated at 90 dpci or with closed compact wall but remaining internal scar or with open compact wall and internal scar remaining. b , 7 dpci: AB, NA, TU n = 7; SAT, WIK n = 6; TL n = 5; KCL n = 3, 21 dpci: AB, SAT, TL, TU n = 5; NA n = 6; WIK n = 7; KCL n = 8 (biological replicates); h , PF-04859989, DMSO n = 6, rotenone n = 5 (biological replicates); i , AB: 1, 90 dpci n = 7; 7 dpci n = 8; 21 dpci n = 6. NA: 1, 7, 90 dpci n = 7; 21 dpci n = 11. SAT: 1, 7, 21, 90 dpci n = 7. TL: 1, 7, 90 dpci n = 7; 21 dpci n = 9. TU: 1, 7 dpci n = 7; 21 dpci n = 5; 90 dpci n = 6. WIK: 1, 7, 21, 90 dpci n = 7. KCL: 1, 7, 21, 90 dpci n = 8 (biological replicates); j , AB, NA, SAT, TL, WIK n = 7; TU n = 6; KCL n = 8. b , h , One-way ANOVA with Tukey’s test. c , d , f , g , Simple linear regression. h , Two-way ANOVA with Tukey’s test. i , Data presented as mean ± s.e.m. Mef2, myocyte enhancer factor 2; PCNA, proliferating cell nuclear antigen.

Article Snippet: Primary antibodies Mef2c (Biorbyt, orb256682), PCNA (clone PC10; Dako, M0879), GFP (Abcam, ab13970), MF20 (Developmental Studies Hybridoma Bank (DSHB), AB_2147781) and embcmhc N2.261 (DSHB, AB_531790) and secondary antibodies Alexa Fluor 488 (Invitrogen, anti-mouse A11001, anti-chick A11039 and anti-rabbit A21206) and Alexa Fluor 555 (Invitrogen, A31570 ) were prepared using TNB buffer at a ratio of 1:200.

Techniques: Immunofluorescence, Staining, Control

Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Gata4, Mef2c, and Tbx5 expression in the beating and non-beating groups. ( a ) Left three panels, gene expression levels in the beating and non-beating group. Right panel, gene expression levels in the beating and non-beating group relative to that of β-actin. ( b ) Representative immunofluorescence images of a cluster of round cells expressing Gata4 and Mef2c on day 14. ( c ) Phase-contrast and immunofluorescence images of beating cells expressing cardiac troponin T (cTnT) and Mef2c on day 28. The arrowhead indicates a beating cell. Scale bar, 100 µm. DAPI, 4′,6-diamidino-2-phenylindole, ** p < 0.01; **** p < 0.0001 by t -test or ANOVA and Tukey’s post hoc test. ( d ) The frequency of round cells expressing Gata4 and Mef2c (10 round cells were examined per experiment, n = 13). ( e ) The frequency of beating cells expressing Mef2c and cTnT (15 beating cells were analysed per experiment, n = 4).

Article Snippet: SVF was transduced with Mouse LentiORF Particles (pLenti-C-mGFP-P2A-Puro; Origene, Rockville, MD, USA): Mef2c (NM_025282, MR226865L4V), Gata4 (NM_008092, MR227022L4V), or Tbx5 (NM_011537, MR227369L4V) for overexpression, or LentiORF Control Particles (pLenti-C-mGFP-P2A-Puro, PS100093V, Origene) as an empty vector control.

Techniques: Expressing, Gene Expression, Immunofluorescence

Journal: Scientific Reports

Article Title: Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

doi: 10.1038/s41598-020-80848-3

Figure Lengend Snippet: Overexpression of SVF with Mef2c augments the number of round cells and their transdifferentiation into CMs. ( a – d ) Phase-contrast images of SVF on day 28 after transfection on day 5 ( a , control; b , Gata4; c , Mef2c; d , Tbx5). Arrowheads indicate round cells; lv, lentivirus vector. ( e ) mRNA level of Mef2c, determined by qRT-PCR, on day 28 in SVFs transfected with the control vector and Mef2c-lv (left), or scrambled shRNA and shMef2c-lv (right), on day 5, respectively. shRNA, short hairpin RNA; shMef2c, shRNA targeting Mef2c. ( f ) Number of round cells on day 14 after introducing SVF with none (none) or the control, Gata4, Mef2c, shMef2c, or Tbx5 lentivirus vector. ( g ) mRNA level of cardiac troponin T by qRT-PCR on day 28 in SVF transfected with the control, Gata4, Mef2c, shMef2c, or Tbx5. ( h ) Relationship between the mRNA levels of Mef2c and cardiac troponin T in the SVF transduced with Mef2c. R, Pearson’s product-moment correlation coefficient. The linear interpolation formula is displayed. Scale bar = 100 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001 by ANOVA and Tukey’s post hoc test.

Article Snippet: SVF was transduced with Mouse LentiORF Particles (pLenti-C-mGFP-P2A-Puro; Origene, Rockville, MD, USA): Mef2c (NM_025282, MR226865L4V), Gata4 (NM_008092, MR227022L4V), or Tbx5 (NM_011537, MR227369L4V) for overexpression, or LentiORF Control Particles (pLenti-C-mGFP-P2A-Puro, PS100093V, Origene) as an empty vector control.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, shRNA, Transduction

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C is a positive regulator of human osteoclastogenesis. a–e Human osteoclast precursor cells were nucleofected with control or two different MEF2C siRNAs. a, b MEF2C mRNA and protein expression. c, d Osteoclastogenesis assay. c TRAP staining of human osteoclasts. Scale bar, 100 μm. d Osteoclast number of three independent experiments. e RT-qPCR analysis of ITGB3 , CTSK , and CTR mRNA after 72 h of culture with or without RANKL (40 ng·mL −1 ) normalized relative to TBP mRNA. Control samples without RANKL were set at 1.0. ( n = 3). f–h Human osteoclast precursor cells were transduced with adenoviral particles encoding GFP or MEF2C-FLAG. f TRAP staining of human osteoclasts transduced with GFP or MEF2C. Scale bar, 100 μm. g Cumulative data showing numbers of osteoclasts from six independent experiments. h Representative images of bone resorption assay of human osteoclasts transduced with GFP or MEF2C. i Quantitation of resorption area from six different experiments. Statistics used: a , d repeated measurement one-way ANOVA e repeated measurement Two-way ANOVA, g , i paired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data were shown as mean ± SD

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Control, Expressing, Staining, Quantitative RT-PCR, Transduction, Quantitation Assay

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C ΔMX mice show increased bone mass with decreased osteoclast numbers. a μCT analysis of femurs from 16-week-old male MEF2C ΔMX KO ( n = 7) and littermate control WT mice ( n = 9). Right panels show the indicated parameters in distal femurs. Bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular numbers (Tb.N), and porosity were determined by μCT analysis. b Histomorphometry analysis of the distal femur of 16-week-old male mice. Representative images showing TRAP-positive, multinucleated osteoclasts (red). Scale bars, 500 μm. Right panels show number of osteoclasts per bone surface (N.Oc/BS), osteoclast surface area per bone surface (Oc.S/BS) and eroded surface per bone surface (ES/BS). c Dynamic bone histomorphometry analysis of the distal femur. Scale bars, 50 μm. Representative images showing casein incorporation into newly calcifying bone. Right panels showed mineral apposition rate (MAR) and bone formation rate (BFR/BS). Data are shown as mean ± SD. Statistics used: a, b, c Welch’s t -test. NS; not significant, * P < 0.05, ** P < 0.01

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Control

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C-deficient cells show impaired osteoclastogenesis. a Representative image of TRAP staining of mouse osteoclasts. Scale bar, 100 μm. b Cumulative data showing numbers of osteoclasts from 6 independent experiments. c RT-qPCR analysis of Itgb3 , Ctsk and Ctr mRNA after 72 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA. Control samples without RANKL were set at 1.0. WT; n = 7, KO; n = 8. Data are shown as mean ± SD. Statistics used: b Welch’s t -test, c Two-way ANOVA. ** P < 0.01, *** P < 0.001

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Staining, Quantitative RT-PCR, Control

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C regulates expression of FOS and c-FOS-target genes in RANKL-stimulated human OCPs. a Heatmap showing relative expression (z-score) of 202 genes differentially expressed with P < 0.01 in RANKL-stimulated MEF2C KD cells versus RANKL-stimulated control cells from three biological replicates. b Enriched transcription factor binding motifs in region ±2 kb relative to transcription start site in RANKL-regulated MEF2C-dependent genes, by gene set enrichment analysis (GSEA). c Volcano plot of RNA-seq analysis of differentially expressed genes in human macrophages transduced with control or MEF2C#2 siRNAs. MEF2C and eight other genes obtained from b are marked. Of note, LEF1 and FOXF2 were filtered out due to low expression level (CPM < 3). Red dots, genes with q value < 0.05 (34 genes). d Cumulative values for FOS in RANKL-stimulated samples from RNA-sequencing with three biological replicates. Data are shown as mean ± SD

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Control, Binding Assay, RNA Sequencing, Transduction

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: c-FOS mRNA and protein expression are dependent on MEF2C in human and mouse OCPs. Human osteoclast precursor cells were nucleofected with control or MEF2C #2 siRNAs. a RT-qPCR analysis of human FOS mRNA after 24 h of culture with or without RANKL normalized relative to TBP mRNA (control samples without RANKL set at 1.0). n = 5. b Representative immunoblotting of c-FOS in nuclear lysates. Lamin B1 and α-tubulin were used as controls for nuclear and cytoplasmic proteins, respectively. c Densitometric quantitation of c-FOS band intensity after 24 h of culture with RANKL from three independent donors. Mouse osteoclast precursor cells from MEF2C ΔMX KO or littermate control WT mice were cultured with M-CSF and RANKL. d RT-qPCR analysis of mouse Fos mRNA after 24 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA (control samples without RANKL set at 1.0). WT; n = 11, KO; n = 12. e Representative images of immunoblotting analysis of c-FOS expression in nuclear lysates. Right panel, densitometric quantitation of band intensity from four samples of each genotype. f Densitometric quantitation of c-FOS band intensity ( n = 4). Human osteoclast precursor cells were transduced with adenoviral particles encoding GFP or MEF2C-FLAG and stimulated with RANKL. g RT-qPCR analysis of human Fos mRNA after 12 h of culture with or without RANKL (40 ng·mL −1 ) normalized relative to TBP mRNA (control samples without RANKL set at 1.0). n = 8. h Immunoblotting of c-FOS at the indicated times. Representative images from five independent experiments. i Densitometric quantitation of c-FOS band intensity from five independent experiments. Data are shown as mean ± SD. Statistics used: a, d, g, i repeated measurement two-way ANOVA, c, f paired t -test. * P < 0.05, ** P < 0.01

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Quantitation Assay, Cell Culture, Transduction

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C binds to the upstream region of FOS gene and regulates NFATC1 expression. a A schematic view of FOS upstream regions with putative MEF2C binding sites predicted based on the analysis of MEF2C ChIP-sequencing data. b ChIP-qPCR analysis of the three putative MEF2C binding sites in the FOS upstream region. MEF2C binding was assessed in human OCPs transduced with adenoviral particles encoding GFP or MEF2C-FLAG and immunoprecipitated with FLAG antibodies. Dotted line represents enrichment level in the negative control HBB region in GFP transduced samples. ( n = 4) Mouse osteoclast precursor cells from MEF2C ΔMX KO or littermate control WT mice were transduced with retroviral particles encoding GFP or FOS and cultured with M-CSF and RANKL. c Representative image of TRAP staining of mouse osteoclasts. Scale bar, 100 μm. d Cumulative data showing numbers of osteoclasts from four independent experiments. Human osteoclast precursor cells were nucleofected with control or MEF2C #2 siRNAs. e RT-qPCR analysis of human NFATC1 mRNA after 48 h of culture with or without RANKL normalized relative to TBP mRNA (control samples with RANKL set at 1.0). n = 5. f Representative immunoblot of human NFATc1. p38 was used as a loading control. Right panel, densitometric quantitation of band intensity from three donors. Mouse osteoclast precursor cells from MEF2C ΔMX mice or littermate control WT mice were cultured with M-CSF and RANKL. g RT-qPCR analysis of mouse Nfatc1 mRNA after 24 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA (control samples without RANKL set at 1.0). WT; n = 11, KO; n = 12. h Representative images of immunoblotting analysis of mouse NFATc1 expression. Right panel, densitometric quantitation of band intensity from 4 samples of each genotype. Data are shown as mean ± SD. Statistics used: e repeated measurement two-way ANOVA, f paired t -test, g two-way ANOVA, h Welch’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Transduction, Immunoprecipitation, Negative Control, Control, Retroviral, Cell Culture, Staining, Quantitative RT-PCR, Western Blot, Quantitation Assay

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C deficient mice show attenuated pathological bone resorption in the K/BxN serum transfer arthritis model. a Schematic of experiments. b Time course of joint swelling and clinical score of K/BxN serum transfer arthritis in WT and MEF2C ΔMX KO mice. WT; n = 5, KO; n = 6. c TRAP staining of histological sections of hind paw and histomorphometric analysis of tarsal bones. Arrow, osteoclasts. Scale bars, 400 μm. Data are shown as mean ± SD. Statistics used: c Welch’s t -test * P < 0.05

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Staining

Journal: Frontiers in Pharmacology

Article Title: Ca 2+ /Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy

doi: 10.3389/fphar.2017.00604

Figure Lengend Snippet: Testosterone-induced MEF2 activity in cardiac myocytes. Cells were transfected with MEF2 luciferase-reporter (MEF2-Luc) and Renilla luciferase plasmids. MEF2 activity is expressed as MEF2-Luc to Renilla luciferase ratio. (A) Cardiac myocytes were stimulated with 100 nM testosterone for 6–48 h ( n = 6). (B) Cells were treated with testosterone at the indicated concentrations for 24 h ( n = 6). IGF-1 treatment (10 nM, 24 h) was used as the positive control for MEF2 activity ( n = 6). (C) Cardiac myocytes were transfected with either siRNA-MEF2C or non-targeting siRNA. (D) Cardiac myocytes expressing MEF2-Luc were transfected with siRNA-MEF2C and stimulated with testosterone (100 nM) for 24 h ( n = 5). Cells transfected with the non-targeting siRNA served as the control. (E) Cells were stimulated with testosterone (100 nM) for 5–180 min and then subjected to immunofluorescent staining with an anti-MEF2C antibody; nuclei were stained with DAPI (blue). The figure shows representative images for control and stimulated conditions (30 min). (F) Quantification of MEF2C staining, shown as the nuclear-to-cytoplasmic fluorescence ratio. Data are presented as means ± SEM or as representative images. P -values were determined using t -test or ANOVA followed by Bonferroni post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control; ### P < 0.001 vs. testosterone.

Article Snippet: The following reagents were from commercial sources: testosterone, bicalutamide, AIP, and 5-bromo-2-deoxyuridine (BrdU), Sigma-Aldrich (St. Louis, MO, United States); anti-phospho-CaMKII (Thr286, cat. # 3361), anti-MEF2C (cat. # 5030) and anti-phospholamban (PLN, cat. # 14562) antibodies, Cell Signaling Technology (Danvers, MA, United States); CellTracker Green (5-chloromethyl fluorescein diacetate) from Thermo-Fisher Scientific (Rockford, IL, United States); anti-phospho-PLN (Thr17, cat. # sc-24565), anti-CaMKII (cat. # sc-5392) and anti-AR (cat. # sc-815) antibodies and siRNAs targeting CaMKIIδ (cat. # sc-38953), AR (cat. # sc-29204), and MEF2C (cat. # sc-38062), Santa Cruz Biotechnology (Santa Cruz, CA, United States); [ 3 H]-leucine, NEN Radiochemicals Perkin Elmer (Waltham, MA, United States); and collagenase type II, Worthington Biochemical Corporation (Lakewood, CA, United States).

Techniques: Activity Assay, Transfection, Luciferase, Positive Control, Expressing, Control, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Ca 2+ /Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy

doi: 10.3389/fphar.2017.00604

Figure Lengend Snippet: Effect of inhibition of CaMKII, MEF2C, and AR on testosterone-induced cardiac myocyte hypertrophy. Cell area and [ 3 H]-leucine incorporation were evaluated as hypertrophy parameters. Cells were treated with 100 nM testosterone for 48 h after (A,B) pretreatment with 1 μM bicalutamide, (C,D) transfection with siRNA-AR, (E,F) pretreatment with 1 μM AIP, or (G,H) transfection with siRNA-MEF2C. Cellular area was assessed using the vital fluorescent dye CellTracker Green ( n = 100 cells per condition from 5 independent cultures). Incorporation of [ 3 H]-leucine was quantified using a liquid scintillation counter, and values are expressed as counts⋅min -1 ⋅(μg of protein) -1 with respect to control non-stimulated conditions ( n = 5). Data are presented as means ± SEM. P -values were determined using ANOVA followed by Bonferroni post hoc test; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. testosterone.

Article Snippet: The following reagents were from commercial sources: testosterone, bicalutamide, AIP, and 5-bromo-2-deoxyuridine (BrdU), Sigma-Aldrich (St. Louis, MO, United States); anti-phospho-CaMKII (Thr286, cat. # 3361), anti-MEF2C (cat. # 5030) and anti-phospholamban (PLN, cat. # 14562) antibodies, Cell Signaling Technology (Danvers, MA, United States); CellTracker Green (5-chloromethyl fluorescein diacetate) from Thermo-Fisher Scientific (Rockford, IL, United States); anti-phospho-PLN (Thr17, cat. # sc-24565), anti-CaMKII (cat. # sc-5392) and anti-AR (cat. # sc-815) antibodies and siRNAs targeting CaMKIIδ (cat. # sc-38953), AR (cat. # sc-29204), and MEF2C (cat. # sc-38062), Santa Cruz Biotechnology (Santa Cruz, CA, United States); [ 3 H]-leucine, NEN Radiochemicals Perkin Elmer (Waltham, MA, United States); and collagenase type II, Worthington Biochemical Corporation (Lakewood, CA, United States).

Techniques: Inhibition, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: Ca 2+ /Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy

doi: 10.3389/fphar.2017.00604

Figure Lengend Snippet: Testosterone increases CaMKII activity and MEF2C and AR protein expression in cardiac hypertrophy in vivo. Extracts of homogenized left-ventricle tissue from the different rat groups were subjected to western blot to measure (A) CaMKII phosphorylation at Thr286 and total CaMKII protein levels, and (B) PLN Thr17 phosphorylation and total protein levels ( n = 6); in these two panels, the values shown are phosphorylated-to-total protein ratios. (C) MEF2C and (D) AR protein levels were determined through western blotting; β-actin was used as the loading control. Data are presented as means ± SEM; P -values were determined using ANOVA followed by Bonferroni post hoc test; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. ORX group.

Article Snippet: The following reagents were from commercial sources: testosterone, bicalutamide, AIP, and 5-bromo-2-deoxyuridine (BrdU), Sigma-Aldrich (St. Louis, MO, United States); anti-phospho-CaMKII (Thr286, cat. # 3361), anti-MEF2C (cat. # 5030) and anti-phospholamban (PLN, cat. # 14562) antibodies, Cell Signaling Technology (Danvers, MA, United States); CellTracker Green (5-chloromethyl fluorescein diacetate) from Thermo-Fisher Scientific (Rockford, IL, United States); anti-phospho-PLN (Thr17, cat. # sc-24565), anti-CaMKII (cat. # sc-5392) and anti-AR (cat. # sc-815) antibodies and siRNAs targeting CaMKIIδ (cat. # sc-38953), AR (cat. # sc-29204), and MEF2C (cat. # sc-38062), Santa Cruz Biotechnology (Santa Cruz, CA, United States); [ 3 H]-leucine, NEN Radiochemicals Perkin Elmer (Waltham, MA, United States); and collagenase type II, Worthington Biochemical Corporation (Lakewood, CA, United States).

Techniques: Activity Assay, Expressing, In Vivo, Western Blot, Phospho-proteomics, Control