FIG 7.

Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H₂O₂ for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H₂O₂ for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H₂O₂ (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P < 0.005 (compared to Syk-EGFP-expressing cells); ***, P < 0.001 (compared to Syk-EGFP-expressing cells). (F) The degree of PARP cleavage was quantified from Western blots of lysates of DG75 cells expressing shRNA for Syk (shSyk), wild-type DG75 cells (WT), or wild-type cells expressing shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H₂O₂ (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. *, P < 0.05 (compared to wild-type cells); **, P < 0.005 (compared to wild-type cells); ***, P < 0.001 (compared to wild-type cells).