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. 2021 Jul 30;11(8):1124. doi: 10.3390/biom11081124

Figure 2.

Figure 2

Ink4a and Arf expression increase in PPCs. ESCs (D0) were induced to differentiate towards PPCs (D4 and D8) and Ink4a and Arf gene expression analyzed by qPCR, Western blot, and immunofluorescence staining. (A) Relative Ink4a and Arf gene expression vs. Gapdh is reported as folds increase. (B) Western blots and densitometric analysis of Ink4a and Arf protein levels relative to Gapdh. (C) Merged images of the immunofluorescence staining at D0 and D8 performed with anti-Ink4a and Arf antibodies (green), rhodamine-phalloidin (for F-actin, red), and DAPI (for DNA, blue). Images were taken with a Zeiss confocal laser-scanning microscope LSM 510 (Oberkochen, Germany), scale bar, 15 μm. (D) Subcellular fractionation of ESCs at D0 and D8: Western blot analysis of equal amounts of cytoplasmic (30 μg) and nuclear (30 μg) protein extracts. Efficiency of cellular fractionation was checked with anti-Parp (for nuclei) and anti-Gapdh (for cytoplasm) antibodies. Normalized Ink4a and Arf band intensities are expressed relative to Gapdh expression in each experimental point. The data are expressed as average of three independent experiments ± SD, * p < 0.05; **, p < 0.01.