Journal: International Journal of Stem Cells
Article Title: p53 Promotes Differentiation of Cardiomyocytes from hiPSC through Wnt Signaling-Mediated Mesendodermal Differentiation
Figure Lengend Snippet: Overexpression of P53 gene in hiPSC by lentivirus methods. (A) Representative images of GFP fluorescence displayed highly efficient infection of hiPSC cells in both P53 and control group. (B) The qRT-PCR results showed that hiPSC with lenti-P53 caused about 8-fold increase of P53 mRNA compared with control group. (C) Representative image of Western Blot showed significantly higher p53 protein level of lenti-P53 group than control, and quantification was done by densitometry (D). (E) The qRT-PCR results showed that overexpressed P53 had no significant effects on the mRNA expression of the pluripotency marker (OCT4, SOX2, NANOG and KLF4) of hiPSC. (F, G) The Western Blot results showed that overexpressed P53 did not change OCT4 protein level but slightly decreased Nanog with statistical significance. Representative image was shown (F) and quantified by densitometry (G). (H) hiPSC with lenti-P53 displayed similar mRNA levels of proliferation markers (MKI67 and AURKB). (I, J) Representative image of cell cycle analysis using flow cytometry showed cell cycle was not influenced by lenti-P53 (I) and quantification by densitometry (J). Expression values of the PCR analysis were normalized to GAPDH. Data are presented as mean±SD (t test, *p
Article Snippet: 48 h later cells were fixed with 4% (w/v) Paraformaldehyde (PFA) for 15 min at room temperature, permeabilized, and incubated with following first primary antibodies: anti-OCT4 antibody (#2750, Cell Signaling Technology, USA), anti-NANOG antibody (#3580, Cell Signaling Technology, USA), anti-SSEA4 antibody (#4755, Cell Signaling Technology, USA), anti-TRA-1-60 antibody (#4746, Cell Signaling Technology, USA), anti-MKI67 antibody (ab15580, abcam, USA), anti-NKX2-5 antibody (ab91196, abcam, USA), anti-cTNT antibody (MS-295-P1, Thermo Fisher Scientific, USA) and anti-α-ACTININ antibody (A7811, Sigma, USA), followed by their corresponding fluorescence-labeled secondary antibodies, alexa fluor 488 labeled goat anti-rabbit IgG (A-11008, Invitrogen, USA), alexa fluor 488 labeled goat anti-mouse IgG (A-11001, Invitrogen, USA), alexa fluor 594 labeled goat anti-rabbit IgG (R37177, Invitrogen, USA) and alexa fluor 594 labeled goat anti-mouse IgG (A-11005, Invitrogen, USA).
Techniques: Over Expression, Fluorescence, Infection, Quantitative RT-PCR, Western Blot, Expressing, Marker, Cell Cycle Assay, Flow Cytometry, Polymerase Chain Reaction