Supplementary Figure 4: In vitro DSB-induced transcription is not dependent on DDR kinases.

(A–C) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α³²P]UTP with pLacTet plasmid (A) or pUC19 plasmid (B,C) and appropriate controls. CFE indicates cell free extract. M indicates radiolabeled DNA ladder, nt indicates nucleotides. (D) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α³²P]UTP with pBluescript plasmid in its circular form or digested with different restriction enzymes generating one DSB with 5’-protruding, blunt or 3’-protruding DNA ends. pLacTet plasmid is used as control. CFE indicates cell free extract. M indicates radiolabeled DNA ladder, nt indicates nucleotides. (E) Agarose gel shows equal amounts of DNA used in D. (F) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α³²P]UTP with pUC19 plasmid in its circular form or digested with different restriction enzymes, as in D. (G–L) Analysis of the role of DDR upstream kinases in the control of transcription from DSBs. Denaturing PAGE showing in vitro transcription assays performed by incubating cell-free extract (CFE) with linear pLacTet plasmid and increasing concentrations (0, 1, 10, 100 nM, 1, 10 μM) of the ATM inhibitor KU60019 (G); with cell-free extract (CFE) prepared from ATM wild-type or knockout embryonic stem (ES) cells (H); with increasing concentrations (0, 1, 10, 100, 1000 μM) of the ATR inhibitor VE-821 (I); with increasing concentrations (0,1, 5, 10, 20, 50 μM) of the PI3K-like kinases inhibitor Wortmannin (J); with increasing concentrations of the following DNA-PKcs inhibitors: (K) NU7441 (0, 10, 100, 1000 nM) and (L) NU7026 (0, 1, 10, 100, 1000 μM). M indicates radiolabeled DNA ladder, nt indicates nucleotides.