Journal: Biology Open

Article Title: Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene

doi: 10.1242/bio.014993

Figure Lengend Snippet: Desmin is a component of transcription factor complexes interacting with regulatory regions of the nkx2.5 gene at the beginning of cardiomyogenesis. (A) Map of the nkx2.5 gene located in negative orientation on mouse chromosome 17 (NC_000083.6; base pair −1 corresponds to position 26.841.565 and A of ATG to 26.841.355 on chromosome 17). The minimal cardiac specific enhancer (MCE) and the proximal enhancer (PE) are indicated as white boxes. Numbers of base pairs at the beginning and end indicate distances relating to the transcription start site. Gray-black boxes, exons; black parts, open reading frame. Pairs of arrows indicate primer binding sites used to amplify DNA obtained by ChIP with desmin specific antibodies. (B) Development of rhythmically contracting CMCs in EBs over time. Bracket indicates the time frame when samples of cells were picked for ChIP analysis at the beginning of cardiomyogenesis. (C) ChIP with desmin-specific antibodies and a PCR primer pair specific for the PE of the nkx2.5 gene in des +/+ , des +/+ des ect , des Δ1-48/Δ1-48 , and des −/− EBs between days 5 and 9 after aggregation. Ip, input DNA before pull-down with antibodies; αD, ChIP with desmin specific antibodies. Genotypes of ESC lines are indicated on the left. Representative PCR data out of five independent biological samples. (D) Representative PCR analysis of same ChIP samples used in C with primer pairs specific for the MCE from days 5 to 8. (E) Mean intensity of the PCR signals (αD) as percentage of the input signal (Ip) from five independent biological samples at regions D2 to P2 along the 5′UTR of the nkx2.5 gene. Error bars, s.d.; * P <0.05. (F) ChIP in differentiating CVPCs and in primary heart cells from newborn mice (CMCs) with desmin-specific antibodies (αD), brachyury-specific antibodies (αT), and IgG (c), as negative control with a PE-specific PCR primer pair.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against desmin (Santa Cruz, sc7559, 1:500, Sigma Aldrich, D1033, 1:50, and Abcam ab8592, 1:80), vimentin (Sigma, V6630, 1:200), cardiac troponin T (Thermo Scientific, MS-295, 1:200, and Santa Cruz, 20025, 1:200), emerin (Novocastra, NCL-Emerin; 1:50), and lamin A/C ( ; 1:500) for 60 min, washed three times with PBS, and stained with FITC- and TR-conjugated secondary antibodies (Dianova, 711-095-152, 1:200; 711-095-151, 1:200; 711-075-152, 1:200), and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, A-11001, 1:1000) for 60 min. Alternatively, cells on cover slips were fixed in 96% ethanol at −20°C for 20 min, dried and blocked with 1% BSA in PBS for 10 min.

Techniques: Binding Assay, Negative Control

Journal: Biology Open

Article Title: Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene

doi: 10.1242/bio.014993

Figure Lengend Snippet: Localization of desmin in nuclei of differentiating cardiac progenitor cells and premature cardiomyocytes. Confocal immunofluorescence micrographs of ESC-derived CPCs at different developmental stages (A-F) and primary premature CMCs (G,H). Desmin (red, A-H), vimentin (green, A-F), and cardiac troponin T (green, G,H) were recognized by specific antibodies. Right images in A,C-E, and images B,F-H are merged images. DNA was counterstained with DAPI (blue). Genotypes of ESC-derived CPCs as indicated. (A) Nuclear localization of desmin in a differentiating ESC-derived CPC at day 5.5. The image is taken from the middle of 20 Z -stack optical sections through the nucleus. (B) 20 Z -stack optical sections of cell shown in A (Step size: 0.5 µm). (C) Undifferentiated des +/+ des ect ESCs. (D) Differentiating des Δ1-48/Δ1-48 ESCs. (E) Differentiating des −/− ESCs. (F) Single des +/+ des ect ESCs with desmin and vimentin positive nucleus. (G,H) Primary immature CMCs from newborn mice. Images 6 out of 12 image stacks. Arrows, intra-nuclear desmin spots. Scale bars: 18 µm.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against desmin (Santa Cruz, sc7559, 1:500, Sigma Aldrich, D1033, 1:50, and Abcam ab8592, 1:80), vimentin (Sigma, V6630, 1:200), cardiac troponin T (Thermo Scientific, MS-295, 1:200, and Santa Cruz, 20025, 1:200), emerin (Novocastra, NCL-Emerin; 1:50), and lamin A/C ( ; 1:500) for 60 min, washed three times with PBS, and stained with FITC- and TR-conjugated secondary antibodies (Dianova, 711-095-152, 1:200; 711-095-151, 1:200; 711-075-152, 1:200), and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, A-11001, 1:1000) for 60 min. Alternatively, cells on cover slips were fixed in 96% ethanol at −20°C for 20 min, dried and blocked with 1% BSA in PBS for 10 min.

Techniques: Immunofluorescence, Derivative Assay

Journal: Biology Open

Article Title: Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene

doi: 10.1242/bio.014993

Figure Lengend Snippet: Desmin and mCherry-tagged desmin partially co-segregate with chromatin in cardiac progenitor cells and primary heart cells and interact with the nkx2.5 gene. (A) Localization of mCherry-desmin in the nucleus. Fluorescence microscopy image of isolated nuclei from mCherry-desmin-expressing ESC-derived CPCs at day 6.7 after aggregation. Scale bar: 3 µm. (B) Quantification of desmin fusion proteins in the nuclei of CPCs. CVPC lines expressing only mCherry, or desmin-mCherry and mCherry-desmin, respectively, were differentiated for 9.3 days. Box plot, median ±25% of data. Whiskers, minimal and maximal values, respectively. Data are from four independent experiments. Numbers of nuclei: mCherry, n =352; Desmin-mCherry, n =360; mCherry-Desmin, n =406. * P <0.0001. (C) Desmin-mCherry and desmin associate with the chromatin fraction. Western blot analysis of cytoplasmic (CP), nucleoplasmic (NP) and chromatin (CT) fractions from desmin-mCherry (rows 1-5) and mCherry only (row 6) expressing CVPC-derived CPCs with antibodies specific for mCherry, desmin, the transmembrane protein gp130, and histone H3. Notably, due to nuclear preparation constraints, the CP fraction contained 1/20 the amount of proteins loaded in the NP and CT fractions. (D) Desmin partially associates with the chromatin fraction in primary immature heart cells from 3-days postpartum mouse hearts. Fractionation of cells and analysis as in C. (E) ChIP analyses with mCherry-specific antibodies in differentiating, mCherry-tagged, desmin-expressing CPCs derived from ESC and CVPC lines (red bars). ChIP signal intensity is depicted as bars on top of the nkx2.5 map highlighting the minimal cardiac specific enhancer (MCE), the proximal enhancer (PE) and the promoter (P), and compared to ChIP data obtained with desmin specific antibodies (black bars) in wild-type cell lines. Pairs of small arrows indicate primer pairs used for PCR analysis. Data are from two independent experiments.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.15% saponin in PBS, and stained with antibodies against desmin (Santa Cruz, sc7559, 1:500, Sigma Aldrich, D1033, 1:50, and Abcam ab8592, 1:80), vimentin (Sigma, V6630, 1:200), cardiac troponin T (Thermo Scientific, MS-295, 1:200, and Santa Cruz, 20025, 1:200), emerin (Novocastra, NCL-Emerin; 1:50), and lamin A/C ( ; 1:500) for 60 min, washed three times with PBS, and stained with FITC- and TR-conjugated secondary antibodies (Dianova, 711-095-152, 1:200; 711-095-151, 1:200; 711-075-152, 1:200), and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, A-11001, 1:1000) for 60 min. Alternatively, cells on cover slips were fixed in 96% ethanol at −20°C for 20 min, dried and blocked with 1% BSA in PBS for 10 min.

Techniques: Fluorescence, Microscopy, Isolation, Expressing, Derivative Assay, Western Blot, Fractionation

Journal: Neural Regeneration Research

Article Title: Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse

doi: 10.3969/j.issn.1673-5374.2012.22.003

Figure Lengend Snippet: Differentiation of adipose-derived stem cells. For myogenisis: (A) immunoflurorescence microscopy revealed that, dko-ADSCs only showed MyoD + cells (12.6 ± 1.5%) and no myotubes. Myotubes appeared on day 28 in dko-microdys-ADSCs (18.0 ± 1.5%) and dko-microdys-β-cat-ADSCs (30.5 ± 2.1%). MHC, desmin, MyoD and myogenin expression in dko-microdys-β-cat-ADSCs was significantly higher than in dko-ADSCs and dko-microdys-ADSCs ( P < 0.05). (B) Reverse trasncription-PCR: the transcription of pax3, pax7, Mif4, Mif5, MHC, myogenin, desmin and MyoD in the two transduced dko-ADSCs groups. For adipogenesis: (C) Oil red O staining. a P < 0.05, vs . dko-ADSCs and dko-microdys-ADSCs. Arrow represents adipogenesis. MyoD: Myogenic differentiation antigen; MHC: myosin heavy chain; ADSCs: adipose-derived stem cells.

Article Snippet: The following primary antibodies were used: MyoD, myogenin, MHC, desmin (MyoD, myogenin, anti-mouse monoclonal antibody; MHC, desmin, rabbit multiple antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-cat (goat multiple antibody, R&D Systems Inc. Basel, Switzerland; 1:200 in PBS), and dystrophin (Abcam; 1:400 in PBS).

Techniques: Derivative Assay, Microscopy, Expressing, Staining