Skip to main content
. 2021 Sep 17;16(9):e0257070. doi: 10.1371/journal.pone.0257070

Fig 4. MiR-200c-3p affects proliferation and regulates senescence through p53, p21 expression in ASCs.

Fig 4

A. Representative images of ASCs transfected with mimic at P4, (low miR-200c-3p) (i) or inhibitor miR-200c-3p at P9, (high miR-200c-3p) (ii) and their corresponding controls. The images were taken at 72 h post-transfection at 40x magnification. B. Clonogenic assays of ASCs transfected with miR-200c-3p mimics (i) and anti-miR-200c (ii) or their respective controls. Cells were stained with crystal violet and subsequently photographed. Absorbance at 595 nm was measured to estimate the optical density in each condition. The black arrows indicate colonies formation. SD± is the mean of optical density of each condition. The experiment was repeated twice and each condition in triplicates and p values were calculated with unpaired t test, ***p<0.001. C. Expression of p-ERK, ERK, Cyclin D1 was studied by WB in ASCs transfected either with mimic at P4 or with anti-miR-200c-3p at P9. On the right, densitometry analysis measures the levels of p-ERK normalized to ERK and Cyclin D1 normalized to β-actin. SD± is the mean of two WBs and the p values were calculated with unpaired t test, **p<0.01. D. (i): The percentage of cells positive for SA-β-galactosidase in ASCs transfected with miRNA mimics or (ii): inhibitors was calculated by counting blue cells in at least 7–8 different areas and in triplicates for each condition. The images (magnification 20x) are representative of one out of three wells. The black arrows indicate β-gal positive cells. SD± is the mean of the % β-gal positive cells detected in three wells for each condition and the p values were calculated with unpaired t test, *p<0.05, **p<0.01. E. Detection of p53, p21 transcripts was performed by qRT-PCR in ASCs transiently transfected at 72 h with (i): mimic-miR-200c-3p, at P4, and the corresponding WBs or (ii): anti-miR-200c-3p, at P9 and the corresponding WBs. GAPDH was used for gene normalization in qRT-PCR and β-actin, loading control for proteins. SD± is the mean of three independent experiments. P values were calculated with two tailed unpaired t test, *p<0.05, ***p<0.001.