Fig 2: Voluntary exercise up-regulates PEDF and suppresses cellular senescence. Six-month-old wild-type mice were subjected to voluntary exercise for 8 weeks. (A) Total RNA was isolated from skeletal muscles (TA and SOL) and Pedf levels were analyzed by real-time PCR. Values were normalized to 18S rRNA in each sample. (B) Serum PEDF levels were analyzed by ELISA. (C) The expression of Ink4a, Arf and p21 in the lungs was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (D) Lung sections were stained for SA-β-gal. Arrowheads indicate SA-β-gal-positive cells. Scale bar, 100 μm. (E) The number of SA-β-gal-positive cells was counted in each sample. (F) The expression of Il-1b, Il-6, and Mmp-12 in lung total RNA was analyzed by real-time PCR. Values were normalized to 18S-rRNA in each sample. Values represent means ± SEM. Data were analyzed by the Student’s t-test. *P <0.05, **P <0.01, and ***P <0.001.

Fig 3: PEDF protected lung tissues from PPE-induced emphysema. (A) Experimental design. A recombinant PEDF protein was injected intraperitoneally twice a week for 4 weeks. PPE (5 units) was intranasally administered 2 weeks after the first dose of PEDF. Mice were analyzed 3 weeks after the PPE treatment. (B) Total RNA was isolated from lung tissues and the expression of Ink4a, Arf, and p21 was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (C) Representative images of control and PEDF-treated mouse lung sections. Sections were stained with hematoxylin and eosin. Scale bar, 100 μm. (D) Alveolar mean linear intercepts were measured. (E–H) A pulmonary function test was performed. Lung compliance (E), capacity (F), tissue damping (G), and tissue elastance (H) are shown. Values represent means ± SEM. Data were analyzed by the Student’s t-test (B), or a one-way ANOVA and Tukey’s post-hoc analysis (D–H). *P <0.05, **P <0.01, and ***P <0.001.

Fig 4: PEDF mediates the anti-cellular senescence effects of C2C12-CM. (A–C) MEFs were cultured in the presence of C2C12-CM treated with a control or PEDF antibody for 3 days. (A) Cell numbers were counted and relative changes in cell numbers in 3 days were plotted. (B) Total RNA was isolated from MEFs and the expression of Ink4a and Arf was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (C) p16INK4a and p19ARF levels were analyzed by immunoblotting. β-Actin was used as the loading control. (D) MEFs were cultured in the presence of a recombinant of PEDF (100 ng/mL) for 3 days. Changes in cell numbers were plotted. (E) Cell viability was determined by the trypan blue exclusion assay. (F) The expression of Ink4a, Arf, and p21 was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (G) p16INK4a, p19ARF, and p21 levels were analyzed by immunoblotting. β-Actin was used as a loading control. (H) Cells were stained for SA-β-gal. Scale bar, 100 μm. (I) The percentage of SA-β-gal-positive cells was plotted. (J) Cells were stimulated with the indicated concentrations of recombinant PEDF for 3 days. Intracellular ROS levels were analyzed in each sample, and relative values were plotted against the average of the control sample. Values represent means ± SD. Data were analyzed by the Student’s t-test (A, B, D–F, I) or a one-way ANOVA and Tukey’s post-hoc analysis (J). *P <0.05, **P <0.01, and ***P <0.001.

Fig 5: Involvement of miR-127 in PEDF signaling. (A) An RNA sequencing analysis was performed on MEFs stimulated with a recombinant PEDF protein. A volcano plot of RNA sequencing data is shown. A full list of RNA sequencing data is available in the Gene Expression Omnibus database (accession number; GSE241459). (B) The expression of miR-127 (-loop, -5p, and -3p) in MEFs was analyzed by real-time PCR. Values were normalized to U6 snRNA in each sample. (C) The expression of BCL-6 in MEFs was analyzed by immunoblotting. β-Actin was used as a loading control. (D, E) miR-127 levels in the lungs of mice subjected to the PEDF treatment (D, Figure 4) or voluntary wheel running (E, Figure 3) were analyzed by real-time PCR. miR-127 levels were normalized to U6 snRNA in each sample. Values represent means ± SD (B) or means ± SEM (D, E). Data were analyzed by the Student’s t-test. *P <0.05, **P <0.01, and ***P <0.001.