![]() Wild type C57BL/6J mice were administered AAV-hTCF21-GFP, AAV-hTCF21-FLAG-YAP, or no virus control. The data correspond to 3 independent cell lines for NP.įigure 2 Characterization of AAV-hTCF21-GFP and AAV-hTCF21-FLAG-YAP expression in mouse hearts. (n = 3 cell culture batches for both), normalized to reference genes, GAPDH and HPRT-1. (G) Relative mRNA expression levels of ACE2 in iPSC-derived cultures NP cultivated for 7 days and CM differentiated for 35 days. (E) Representative flow cytometry graph showing a quantification of 83.2% TNNT2 + cells from one of the cell batches used in this study (F) CM immunostained for cardiac troponin T (TNNT2). NP were immunostained for (B) Nestin (neural progenitors), (C) S100b, (astrocytes) and (D) MAP-2 (neurons). NP were cultivated for 7 days and CM were differentiated for 35 days and characterized by IF (B-D and F) and flow cytometry (E). Consensus normalized expression (NX) and z-score are used to represent the relative abundances within the respective databases (Citations in the main text). (A) ACE2 expression in adult human tissues based on Protein Atlas and Allen Brain Atlas databases. 1 ACE2 expression in adult human tissues and iPS-derived cells. aCD47, anti-CD47 neutralizing antibody Dox, doxorubicin p-, phosphorylated cTnT, cardiac troponin T.įig. Two-way ANOVA followed by a Tukey's multiple comparison's test. Bar plot data in all panels are represented as mean +- standard error. (E) Association between the percentage of p-p38 MAPK + cells and apoptotic cardiomyocytes after treatment. Data are presented as the percentage of p-p38 MAPK + cells. (D) p-p38 MAPK + cells after Dox and aCD47 treatment were analyzed by flow cytometry. (C) Western blot analysis for the expression of pro-caspase-1, cleaved caspase-1, cleaved caspase-3, Bax and Bcl-2 in the treated cardiomyocytes. Data are presented as the relative intensity of positively stained cells over untreated controls. (B) Semi-quantitative analysis of positively stained cells by ImageJ software. Representative images with x200 magnification. Red, cells positively stained for cleaved caspase-1/3, Bax and p-p38 MAPK. Cardiomyocytes were identified as cTnT-positive cells (green). (A) Immunostaining for the expression of cTnT, cleaved caspase-1/3, Bax and p-p38 MAPK in the treated cardiomyocytes. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).įigure 5 aCD47 reduces the expression of Bax and p-p38 MAPK in Dox-treated cardiomyocytes. ![]() The membrane was probed with the relevant primary antibody following blocking with 5 % skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). A 40 kDa band corresponding to Cardiac Troponin T was observed across the tissue extracts tested. The blots were probed with Anti-Cardiac Troponin T Monoclonal Antibody (Product # MA5-12960, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Western blot analysis was performed on tissue extracts (30 µg lysate) of Mouse lung (Lane 1), Rat lung (Lane 2), Mouse skeletal muscle (Lane 3), Rat heart (Lane 4) and Mouse heart (Lane 5). Images were taken at 20X (Panels A and B) or 60X (Panel C) magnifications on a Carl Zeiss microscope (AxioVision Rel. Nuclei (blue) were visualized using Hoechst 33342 (Product # 62249) and dead cells were visualized using Propidium iodide (red). Cells were probed with a Troponin T Cardiac Isoform monoclonal antibody (Product # MA5-12960) at dilution of 1:300 for 2 hours at room temperature or overnight at 4 ☌, washed with HBSS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at dilution of 1:500 for 1 hour at room temperature. At day 1 and day 7, cells were fixed with 4% paraformaldehyde, permeablilized with 0.1% Triton X-100 in HBSS for 10 minutes at room temperature, and blocked with 3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were grown in 24-well plates (seeded at 5x10^5 cells/well) or in 35mm glass bottom plates (at a density of 2.5 x 106 cells/well). Primary cardiomyocytes were isolated and cultured using the Primary Cardiomyocyte Isolation Kit (Product # 88281). Immunofluorescent analysis of Troponin T Cardiac Isoform (green) in cultured primary cardiomyocytes.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |