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. 2023 Jul 4;26(8):107283. doi: 10.1016/j.isci.2023.107283

Figure 2.

Figure 2

The impact of cardiac ECM on Kindlin-2 expression

(A) Representative immunostaining of Kindlin-2 (red) and α-SMA (green) for tridimensional (3-D) culture of cardiac fibroblasts seeded on cardiac matrices from age-matched or un-matched mouse donors for 72 h. Representative pictures from 3 to 4 experiments per group (young vs. old) and two donors (young vs. old) per experiment. Note that the old male matrices tend to exhibit a more intense autofluorescence. Scale bar: 20 μm.

(B) Immunostaining for α-SMA (green) and F-actin (red) in old male cardiac fibroblasts seeded on glass (left), on matrix from old male cells (middle) or matrix from young male cells (right) (scale bar: 20 μm). Nuclei were counterstained with DAPI.

(C) Representative Western blot of Kindlin-2 expressed by cells seeded on age-matched and unmatched matrices in the 2-D experiment. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 5–6 per group).

(D) Representative Western blot showing the level of Kindlin-2 expressed by old male and old female fibroblasts seeded on sex-matched or unmatched matrices. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 4–5 per group). YM, OM, OF denote the young male, old male, and old female, respectively. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot.

(E) Scanning electron microscopy pictures of decellularized 3-D matrices from young or old mouse male hearts (upper panel), young or old female hearts (middle panel) or decellularized 2-D matrices deposited by young or old male cardiac fibroblasts (lower panel). Red arrows indicate cleavage sites in the old male cardiac ECM, and cyan circles depict aggregates in the old female cardiac ECM. Scale bar: 2 μm. Pictures in enlarged frames (yellow squares) depict the presence of cleavage in the old male ECM and aggregates in the old female ECM. All the data are shown as an averaged mean ± SEM. For the experimental dataset involving 4 groups, Brown-Forsythe and Welch one-way ANOVA was used to test the significant difference among the four groups, to determine the significant difference among groups. p value <0.05 is considered statistically significant.