PPAR accelerates cellular senescence by inducing p16^INK4 expression in human diploid fibroblasts

JCS026633 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1AB
• Supplemental Figure S1C -

  Fig. S1. Ligand-activated PPARγ induced changes associated with senescence and irreversible cell-growth arrest in 2BS. 2BS cells transfected with the expression plasmids pcDNA3.1 (vector) or pcDNA-PPARγ (PPARγ) were analyzed for the relative senescence markers (all at PD42). Cells were treated with 10 µM pioglitazone or DMSO (vehicle) as indicated. Untransfected young (Y: PD25), middle-aged (M: PD42) and senescent (S: PD62) 2BS cells were also analyzed for comparison. (A) Young, middle-aged and senescent 2BS cells and vector-transfected or PPARγ-transfected cells were stained for SA-β-gal activity, a classical marker of senescence. (B) Growth curves of vector-transfected or PPARγ-transfected young, middle-aged and senescent 2BS cells were determined by the MTT assay. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. Values in B followed by different symbols are statistically significantly different from each other. (C) Flow-cytometry analysis of vector-transfected, PPARγ-transfected, young, middle-aged and senescent 2BS cells. Each experiment was performed at least three times. The table shows the representative data. The graph depicts data from three independent experiments (means ± s.d.). *P&λτ;0.05 vs vehicle-treated vector cells. #P&λτ;0.001 vs vehicle-treated vector cells; &P&λτ;0.05 vs untransfected young 2BS cells. †P&λτ;0.001 vs untransfected young 2BS cells.

• Supplemental Figure S2 -

  Fig. S2. PPARγ activation increases p16 protein levels in 2BS cells. 2BS cells were transfected with the expression plasmids pSilencer 2.1-U6 neo (RNAi vector), pSilencer-PPARγ (siPPARγ) or pSilencer-negative control siRNA (negative) and treated with 20 µM troglitazone or DMSO (vehicle) as indicated for 72 hours. (A) Western blot analysis of PPARγ, PPARα and PPARβ expression in RNAi-vector-transfected, negative-transfected or PPARγ-transfected cells. (B) Western blot analysis of PPARγ and p16 expression in negative-transfected or siPPARγ-transfected cells. Western blotting was performed using specific antibodies against PPARγ, PPARα, PPARγ and p16^INK4α as indicated. The β-actin lane served as loading control.