FIG. 7.

Transfection of ESC with a Geminin short hairpin RNA (shRNA) plasmid causes DNA damage. (A) Diagrams of a control plasmid containing 3 nucleotide substitutions producing a mutated shRNA and the shRNA targeting Geminin. (B) Western blot of protein from ESC transfected for 24h with mutated shRNA or Geminin shRNA, demonstrating a 6-fold reduction in Geminin protein. (C) ESC were transfected with mutated (a, c, e) or Geminin shRNA (b, d, f). 11.4%±0.6% of cells exposed to the control (mutated) shRNA express phosphohistone H2A.X (an indicator of DNA damage, arrows), whereas the loss of Geminin protein induced widespread DNA damage (84.3%±0.1% of transfected cells, P≤0.001). Scale bar=200 μM. Color images available online at www.liebertpub.com/scd