(A) NEs isolated from the mutant SH-SY5Y cells (NT-mHTT-Q97) (lane 2) and mutant cells with PNKP overexpression (lane 3), and PNKP activity was measured. No protein was added in lane 1 (NP) and purified PNKP was added in lane 4 (PP). DNA substrate and the released phosphate are shown by arrows. (B) Alkaline comet analyses of cells expressing NT-wtHTT-Q23 (left panel), NT-mHTT-Q97 (center panel), and co-expressing NT-mHTT-Q97 and PNKP (right panel). Comet tails (DNA strand breaks/damages) are shown by arrows. Nuclei are stained with SYBR green. (C) Tail moments representing strand breaks in cells expressing NT-wtHTT-Q23, NT-mHTT-Q97, and co-expressing NT-mHTT-Q97 and PNKP. Data represent mean ± SEM; *p<0.001 compared with NT-wtHTTQ23 and # p<0.001 compared with NT-mHTT-Q97 in C, E, G and I. (D) Expression of NT-mHTT-Q97 was induced (by adding doxycycline to the medium) in differentiated SH-SY5Y cells that were harvested 0, 48, and 72 hr post-induction, and NEs were analyzed by WBs to detect p-ATM-S1981, total ATM, p-p53-S15, total p53, p-γH2AX-S139, and total H2A levels; β-tubulin is the loading control. (E) Expression of NT-mHTT-Q97 was induced in differentiated SH-SY5Y cells overexpressing PNKP. NEs were analyzed to determine the levels of p-ATM-S1981, total ATM, p-γH2AX-S139, total H2A, p-p53-S15, and total p53; β-tubulin is the loading control. (F) Relative levels of p-ATM-S1981, p-γH2AX, and p-p53-S15 with respect to the corresponding total proteins. Cells were harvested 0 (gray), 48 (red), and 72 (blue) h after NT-mHTT-Q97 expression; Data represent mean ± SEM. (G) Caspase-3 activities in PC12 cells expressing FL-wtHTT-Q23, FL-mHTT-Q148, co-expressing FL-wtHTT-Q23 and PNKP, or co-expressing FL-mHTT-Q148 and PNKP. (H) Representative flow cytometry analysis showing cell toxicity in PC12 cells expressing FL-wtHTT-Q23, FL-mHTT-Q148, and co-expressing FL-mHTT-Q148 + PNKP. X-axis: Annexin-V-FITC signal. Y-axis: Propidium Iodide signal. Healthy cells are represented in the lower left quadrant, early apoptotic cells in the lower right quadrant, late apoptotic cells in the upper right quadrant, and necrotic cells in the upper left quadrant. (I) Relative levels of cell toxicity of cells expressing FL-wtHTT-Q23, FL-mHTT-Q148 and co-expressing FL-mHTT-Q148 and PNKP. Data represent mean ± SD; *p<0.001 when compared wtHTT-Q23 vs. mHTT-Q148, and #p<0.001 when compared mHTT-Q148 +PNKP with mHTT-Q148.