(A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) pretreated with Olaparib (10 μM), PJ-34 (10 μM), or DMSO for 1 hr, were stimulated with 10 μM of CPT for indicated periods, and whole cell lysates were immunoblotted (IB) for indicated proteins, with β-actin as a loading control. (B and C) Primary colonic epithelial cells (CECs) isolated from Khdrbs1+/- and Khdrbs1-/- mice were stimulated with 10 μM of CPT (B) or 10 Gy of γ-irradiation (IR) (C). Whole cell lysates were derived at indicated periods post stimulation and IB as in (A). Right, the PAR levels, normalized to β-actin and untreated controls, were quantified from three independent experiments. (D) Immunofluorescence micrographs of PARylated proteins (PAR) in CECs treated as in (C), with nuclei counterstained by DAPI. Scale bar, 10 μm. Percentage of CECs (>100 cells from 5–8 random fields) with PAR staining was quantified (right). (E) WT and Sam68 KO MEFs were γ-irradiated (IR) at 10 Gy and the chromatin, soluble (Sol. fr.), and insoluble (Ins. fr.) subcellular fractions were derived at indicated time points post γ-irradiation and IB for Sam68, Caspase-3 (Casp-3), and Histone H3 (H3). (F) WT MEFs were stimulated with 10 μM of CPT for indicated periods and the chromatin fractions were derived and IB as in (E). Right, the Sam68 and PARP1 levels in the chromatin fractions, normalized to H3 and untreated controls, were quantified from three independent experiments. (G) Whole cell lysates from WT and Sam68 KO MEFs stimulated with 10 μM of CPT for 20 min, were IB directly or after immunoprecipitation (IP) with PARP1 antibody for indicated proteins. (H) Recombinant PARP1 protein was incubated in reaction buffer containing damaged DNA or with purified GST or GST-Sam68 protein in the presence or absence of NAD+ or the PARP1 inhibitor PJ-34, as indicated. The reaction mixture was separated by SDS-PAGE and subjected to IB with the PAR, PARP1, and Sam68 antibodies. Results in (B, C, D and F) are expressed as mean and s.e.m. ns, non-significant difference and , *p<0.05, ***p<0.001 by Student’s t tests. Data are representative of at least three independent experiments.