Figure 1. PTP1B oxidation was required for H-RAS^V12 induced senescence.

(A) Representative photographs of IMR90 fibroblasts stained for SA β-gal activity. (B) H-RAS^V12-induced H₂O₂ production was assessed by molecular imaging using PF6-AM. (C) Levels of H₂O₂ released in the culture medium of H-RAS^V12-transformed cells were quantitated by Amplex Red. (D) Control or H-RAS^V12-induced senescent cells were subjected to the cysteinyl-labeling assay, using biotinylated IAP at pH 5.5. Biotinylated proteins were purified on streptavidin–Sepharose beads, resolved by SDS-PAGE, and visualized using anti-Biotin-HRP. (E) Proteins enriched in the cysteinyl-labeling assay were resolved by SDS-PAGE and immunoblotted using an anti-PTP1B antibody (FG6) (upper panel). Total PTP1B and H-RAS^V12 expression levels were measured by immunoblotting 5% of the lysate. (F) Control or H-RAS^V12-induced senescent cells were cultured with or without 1 mM N-acetylcysteine (NAC) for 2, 4 or 6 days, then subjected to the cysteinyl-labeling assay. Reversibly oxidized PTP1B was visualized using FG6. Total H-RAS^V12 and p21 expression levels were measured by immunoblotting 5% of the lysate. (G) Cells infected with H-RAS^V12 and PTP1B (WT) or a catalytically impaired PTP1B mutant [PTP1B (CS)] were stained for β-gal following a 2 day puromycin selection (H-RAS^V12) and 6 day hygromycin selection (PTP1B). (H) Control (white bars) or H-RAS^V12-expressing cells (black bars) were cultured in the presence or absence of the PTP1B inhibitor, MSI-1436 (20 µM). Cells were stained for β-gal activity 2 days post-puromycin selection. (I) Control (white bars) or H-RAS^V12-expressing cells (black bars) were cultured in the absence or in the presence of 1 mM NAC, 20 µM MSI-1436, or both, for a 6 day-period post-selection. In samples stained for β-gal activity, cells were counted and expressed as the percentage of senescent cells in the total population. Error bars represent SEM (n=3).