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
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