Detailed Immunostaining and Transfection Protocols

Sources of chemicals were as previously described^{15 (link),19 (link),30 (link)}. The following antibodies were used at the indicated dilutions for immunofluorescence (IF) or immunoblot (IB): antibodies against γH2AX and H2AX (1:500 (IF) and 1:1000 (IB)) were from Cell Signaling; the antibody against the cytosolic tail of L2A (1:100 (IF) and 1:5,000 (IB)) was from Invitrogen, against LAMP1 (1:100 (IF) and 1:1,000 (IB)) was from Iowa University Hybridoma Bank, against hsc70 (13D3, 1:1,000) from Novus Biotechnology, against GAPDH (1,5000) and actin (1:5,000) from Abcam, against GFP (1:100 (IF) and 1:3,000 (IB)) from Roche, against p62 (1:3,000 (IB)) from Biomol international, against pChk1S345, pChk1S317, Chk1, Chk2, Mre11, Nbs1, Rad50, ATM and ATR (1:1,000 (IB), and 1:100 (IF)) from Cell Signaling. LC3 antibody was from cell Signaling, used at 1:2,000 (IB). Lamin A/C antibody was from abcam, used at 1:3,000 (IB). The plasmid for Chk1 (peGFP-Chk1) was from Addgene and the plasmid for human L2A (pGK-hL2A) was generated in our laboratory^{31 (link)}. Cells were transfected with cDNA constructs using Lipofectamine™ 2000 reagent (Invitrogen) according to manufacturer’s instructions. Transfection efficiency was monitorized by co-expression of a different fluorescent protein. Mutagenesis was performed using the Quikchange II Site-directed mutagenesis kit (Agilent Technologies).