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. 2019 Apr 30;12(2):251–264. doi: 10.15283/ijsc18126

Fig. 3.

Fig. 3

Evaluation of Connexin43 (Cx43) and Nkx2.5 levels in induced AF-MSCs. (A) The levels of Cx43, the main gap junction protein, and Nkx2.5, an early cardiac transcription factor, as determined by Western blot in non-differentiated cells (Ctrl) and differentiated cells at the days 5 and 12. As a positive control mouse heart lysate was used. The relative density of each band was measured using ImageJ software (NIH, USA), normalized to the GAPDH loading control and presented as a fold difference over control. The data are presented as mean±SD (n=3), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), ns: non-significant. The blots represent one of three independent experiments showing similar results. (B) The distribution of Cx43 in control and induced cells at the 12 day of differentiation as obtained using the immunofluorescence assay. The data from each differentiation sample is presented in two images: the left panel–two channels view (blue nuclei and green Cx43) and the right panel–the same image in three channels view (blue nuclei, green Cx43 and red F-actin). Samples were observed using Zeiss Axio Observer fluorescence microscope, 63X magnification with immersion oil, scale bar=10 μm. (C) The immunofluorescence data of Nkx2.5 protein in control and induced cells at the 12 day of differentiation. Nuclei stained blue, Nkx2.5 – red and F-actin – green. Samples were observed using Zeiss Axio Observer fluorescence microscope, 63X magnification with immersion oil, scale bar=10 μm. (D) Quantitative evaluation of Nkx2.5 nuclear localization at the day 12 of differentiation as calculated from the Nkx2.5 immunofluorescence images. The nuclear quantity of Nkx2.5 was normalized to the median value of undifferentiated Control (Ctrl) and presented as normalized nuclear quantity (NNQ). All NNQs were graphed as Tukey-style box plots with sample size n=66, 31, 57, 58, 64 and 60. Relative change of medians and the statistical significance of changes are presented above the box plots. Wilcoxon rank sum test was used for statistical analysis, p≤0.001 (***).