Figure 3. Cyclophilin B suppresses cellular senescence.

(A) Representative gene expression changes in CypB-depleted U251 cells. (B) Increased IL-11 (left) or decreased GFAP (right) mRNA levels in CypB-depleted U251 cells were confirmed by real-time PCR. (C) Increased SERPINE1 and decreased SERPINA3 expression were confirmed by westernblotting in CypB-depleted U251 cells. (D) Cross match analysis between microarray results from CypB silencing and those from oncogenic H-Ras-activated senescent cells. (E) Morphological changes in CypB-depleted U251 cells. CypB-knockdown cells had cytoplasmic vacuolization and large, flat appearance. Bar=200µm. (F and G) Morphological features (F) and molecular changes (G) of oncogenic H-Ras-expressed GBM cells. U251 cells were transduced with a lentivirus expressing oncogenic H-Ras (HRASV12) or control lentivirus (Vector) and cell morphology was examined by microscopy (F). Ras expression, subsequent Erk activation, and expression changes in SERPINE1 and SERPINA3 were measured by westernblotting (G). Bar=200µm. (H) Increased positive staining of the senescence-associated β-galactosidase marker in CypB-silenced U87 cells. Bar=200µm. (I) Increased Erk activation and p27 accumulation in CypB-depleted U251 cells. Cells were transduced with shCypB (48 or 49) or control lentivirus (C), and analyzed at days 3, 4, and 5. (J) Increased Ras activation in CypB-depleted U251 cells. Active form of Ras (RAS-GTP) was pulled down with Raf-1 RBD (Millipore) and detected by westernblot. (K and L) Increased expression of p27 in CypB-depleted T98G (K) or U87 cells (L). (M) CsA treatment (20 µM) reduced Skp2 expression in U251 cells. Errorbars show ±SD. *p<0.05.