Figure 2.
Metnase promotes progression through the metaphase decatenation checkpoint. (A) Manipulated Metnase levels in HEK-293 and HEK-293T cells examined using western analysis with anti-Metnase antibody and RT–PCR as described (5). β-Actin and 18S rRNA served as loading controls for western blots and RT–PCR, respectively. HEK-293, but not HEK-293T, cells express endogenous Metnase. Stable Metnase over-expression was achieved in HEK-293 cells transfected with pCD-Metnase, and HEK-293T cells transfected with pCAPP-Metnase; siMetnase indicates stably transfected siRNA Metnase knockdown in HEK-293. (B) Decatenation checkpoint arrest induced by ICRF-193 was monitored by the increase in metaphase cells using immunofluorescence microscopy of cells stained with anti-tubulin antibodies (green) and DAPI (blue). pCD-vector control, U6-siRNA control. (C) Quantitative analysis of ICRF-193 or VP-16 induces mitotic checkpoint arrest after subtraction of vehicle controls. (D) HEK-293T cells stably transfected with pCAPP-Metnase or empty vector were seeded in medium with or without 0.5 μM ICRF-193 on Day 0 and counted daily. HEK-293T cells stably transfected with pCAPP-Metnase or empty vector were seeded in medium with or without 5 μM VP-16 on Day 0 and counted daily thereafter. (E) Flow cytometric analysis of G2/M fractions in HEK-293T cells stably transfected with empty or Metnase expression vectors after 10 µM ICRF-193 treatment.