(A) Control and ARH3 KO U2OS (ARH3-/-) cells were treated with 2 mM H2O2 for the indicated time points. After treatment, cells were lysed and proteins were separated by SDS-PAGE, analysed by western blot and probed for PAR, pan-ADPr, ARH3, H3, and GAPDH antibodies. Additionally, Ponceau-S staining was used as loading control (B) Cell extracts obtained from ARH3 KO cells treated with 2 mM H2O2 for 10 and 120 min were incubated with buffer or ADP-ribosylhydrolases ARH3, MACROD2 and PARG. Samples were separated by SDS-PAGE, analysed by Western blot and probed for pan-ADPr, H3, GAPDH, and Ponceau-S staining were used as loading control. (C) Schematic representation of the SILAC-based strategy to quantify core histone Ser-ADPr marks from Control U2OS cells (top panel) or ARH3 KO U2OS cells (bottom panel) after H2O2 treatment for 120 min. Heavy labeled (Lys8) Control U2OS cells treated for 10 min with H2O2 were used as SILAC Standard. (D) MS1s of a Ser-ADPr H2B peptide (H2B Ser6-ADPr) from Control U2OS cells (top panel) or ARH3 KO U2OS cells (bottom panel) after H2O2-treatment for 120 min. The light peptide was derived from Control (top panel) or ARH3 KO (bottom panel) cells treated with 2 mM H2O2 for 120 min, and the heavy peptide was derived from the SILAC Standard (10 min H2O2 treated cells). Each inset shows a ∼1:1 ratio (heavy/light) of a non-ADP-ribosylated peptide from the same experiment. In the ARH3 KO U2OS cells, the chosen H2B mark (H2B S6-ADPr) resulted ~40 times more abundant than in the Control U2OS cells.