Figure 1.

The aging phenotype of the male cardiac fibroblast is associated with a decreased expression of Kindlin-2

(A) Representative Western blot depicting the difference in α-SMA protein level between the young and old male cardiac fibroblasts (upper panel) and the diagram representing the quantification from separated experiments (N = 5–6 per group) (lower panel).

(B) Representative Western blot for the actin (“F” depicting F-actin/Polymerized, and “G” for globular/monomeric). Actin quantity was estimated by densitometry for each sample, and the final F-to-G-actin ratio is shown in an individual-plotted diagram on the lower panel (N = 9–10 per group).

(C) Representative F-actin staining for young and old male cardiac fibroblasts seeded on glass, scale bar: 20 μm. N = 10 individual samples from 4 separate experiments.

(D) Representative Western blot depicting the difference in Kindlin-2 protein level in the lysate from young versus old cultured cardiac fibroblasts. Normalized densitometry was shown on the lower panel as an averaged value of 6–8 separated experiments.

(E) Staining for Kindlin-2 (green) with a DAPI nucleus counterstaining (blue) for young and old cardiac fibroblasts, scale bar: 20 μm.

(F–H) Representative staining for F-actin (F), α-SMA (G) and Kindlin-2/α-SMA (H) in cultured old male and old female cardiac fibroblasts, seeded on glass. Representative pictures from 3 to 4 biological repeats. Scale bar: 20 μm.

(I) Western blot depicting the F-/G-actin fractions in the lysate from old female versus old male cardiac fibroblasts. The diagram (lower panel) shows the relative comparison of the F-to-G-actin. N = 4.

(J) Western blot demonstrating the level of Kindlin-2 and α-SMA in the lysates from young and old female cardiac fibroblasts. Normalized densitometry is depicted as an individual-dotted diagram for both Kindlin-2 (lower left) and α-SMA (lower right). P-value was shown with an "N.S." below the bar, indicating a non-significant difference. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot.

(K) Dot-plots depicting the correlation between the level of Kindlin-2 or F-/G-actin ratio (y axis) versus α-SMA (x axis). The regression line shows a positive correlation in male cells (left panel) but not in female cells (middle panel). There is also a positive correlation between the F-/G-actin ratio and the level of α-SMA (right panel). Statistics: All the data are shown as an averaged mean ± SEM. P-value was calculated as an Unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.