SENP6 knockdown induces a hyper-acute apoptotic response in MSH2-deficient human cells. (A-D) Efficient targeted knockdown of SENP6 or SENP7 in MSH2-deficient HEC59 cells. In A, B mRNA was quantified by qPCR; in C, D SENP6 protein was detected by flow cytometry (see Methods). Note specificity of SENP6 knockdown in response to siSENP6-C or siSENP6-1, but not siSENP7 (pooled siSENP7-1 and siSENP7-2) or non-targeted siNT-1. (E) SENP6 (but not SENP7) knockdown induces apoptosis in HEC59 cells (MSH2") relative to an isogenic chromosome 2 complemented population (MSH2+). Caspase 3+ cells were identified 48h after siRNA transfection (see Methods), normalized to siNT-1 in each population, and significance determined by unpaired t-test (ns, not significant; *, P <0.01). (F) PARP inhibition induces DNA DSBs (γH2AX+), and to a greater degree in MSH2− cells. γH2AX+ cells were identified 72h after siRNA transfection/24h after PARP inhibition (20μM olaparib; see Methods) and the significance of pairwise comparisons determined by unpaired t-test: (1) P <10−4; (2) ns, not significant; (3) P <0.008; (4) P <0.002. Panels (F-H) use the same color key and siSENP6-1 for SENP6 knockdown (previous studies suggest off-target effects are unlikely ). (G) MSH2− cells show reduced clonogenic survival (see Methods) in response to SENP6 knockdown, PARP inhibition, or their combination. Any significance of indicated pairwise combinations was determined by unpaired t-test: (1) P <0.02 (may be related to the additional copy of chromosome 2); (2) ns, not significant; (3) P <0.001; (4) P <0.01; (5) P <0.0007; (6) P <0.03; (7) P <0.01. (H) (MSH2/SENP6) deficient cells exhibit a hyper-apoptotic response to PARP inhibition. Activated caspase 3 levels 72h after siRNA transfection/24h after PARP inhibition (20μM olaparib) were quantified by flow cytometry and normalized to siNT-1 (no drug). Any significance of various pairwise combinations was determined by unpaired t-test: (1) P <10−5; (2) P <10−6 (also seen with MSH3−); (3) P <10−7.