FIG 6.

MEKK1 contributes to damage-induced degradation of p21 and DDB2 and cell survival. (A) HEK293 cells were transfected to express plasmids encoding specific shRNAs targeting ROC1, MEKK1, or a scrambled control and selected for 5 days with puromycin. Subsequently, cells were treated for various periods with 4NQO, and expression of the indicated proteins was revealed by immunoblotting. (B) HEK293 cells were transfected to express MEKK1-specific or control shRNAs and selected for 5 days with puromycin. Subsequently, cells were reseeded and transfected as shown with shRNA-resistant forms of MEKK1 and its mutants, followed by 4NQO treatment and Western blotting to detect p21 degradation (top) and MEKK1 knockdown (bottom). (C) Schematic summary of the results. (D) HeLa cells allowing the Dox-inducible expression of miR30-based shRNA for MEKK1 were generated (HeLa pIND-MEKK1) and treated for 4 days with Dox. Cells were then exposed for the indicated periods to 4NQO (to induce DNA damage) and MG132 (to prevent degradation of DDBs), followed by fractionation of cells into cytosolic, nuclear, and chromatin fractions. These fractions were analyzed for the kinetics of inducible CUL4A and DDB2 import into the nucleus and chromatin. The purity of the fractions was controlled by blotting for tubulin (cytosol), PARP (nuclear soluble), and histone H3 (chromatin). The relative levels of CUL4A in the various fractions was quantified using the ChemiDoc imaging system, and relative protein amounts were normalized to the marker protein of the respective fraction. Maximal expression in each fraction was arbitrarily set to 1; the median values are indicated. (E) HeLa pIND-MEKK1 cells were treated with Dox and/or 4NQO as shown. While cells from two dishes were lysed 4 days after Dox treatment and further tested for efficient MEKK1 knockdown, dishes containing the other cells were washed, and cells were further grown to form colonies for 1 week. Cells were stained with crystal violet. Representative results are shown.