Sc 5221

The cells were transfected with Setdb1 siRNA for 48 h. Approximately 30 μg protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were probed using the following primary antibodies: NOX4 (1:500; NB110-58849; Novus), beta-Actin (1:2000; CW0096; CWBIO), SETDB1 (1:1000; 11231-1-AP; Proteintech), E2F1 (1:500; sc-193; Santa Cruz Biotechnology), FOXO4 (1:500; sc-5221; Santa Cruz Biotechnology), Lamin B (1:500; sc-6217; Santa Cruz Biotechnology), JNK (1:500; sc-7345; Santa Cruz Biotechnology), p-JNK (1:400; WL01813; WanleiBio), γH2AX (1:000; 2577; Cell signaling technology), p38 (1:500; sc-7972; Santa Cruz Biotechnology), and p-P38 (1:500; sc-17852-R; Santa Cruz Biotechnology). All were used as the manufacturer’s recommendation. The secondary antibodies were horse radish peroxidase-linked anti-mouse, anti-rabbit, or anti-goat IgG for 2 h at room temperature. The membranes were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Bio-Rad, Berkeley, CA, United States).

The cells were seeded onto a 96-well plate and transfected with siRNA for 48 h. The cells were fixed with 4% PFA for 30 min, permeabilized in 0.5% TritonX-100 for 10 min, and blocked in 3% BSA for 2 h. The cells were incubated with primary antibody for FOXO4 (sc-5221; Santa Cruz Biotechnology) and γH2AX (2577; Cell signaling technology) overnight at 4°C. After washing with PBS, the cells were incubated for 1 h with secondary antibody, followed by incubation with DAPI.

The ChIP experiment was performed using Active Motif’s ChIP assay kit (catalog 53035) as we described previously (33 (link)), with anti-FoxO4 antibody (sc-5221, Santa Cruz Biotechnology) or rabbit IgG (ab37415, Abcam). The precipitated DNA was quantified by real-time qPCR with a Bio-Rad CFX96 real-time system using the primer specific for different targets. Primers for FoxO4 ChIP-qPCR were synthesized as reported previously (10 (link)).
For ChIP-Seq, input and endogenous FoxO4 ChIPed DNA obtained by the ChIP procedure above were subjected to library preparation using the NEXTflex ChIP-Seq DNA Sequencing Kit (5143, Bioo Scientific). The DNA libraries were sequenced on an Illumina HiSeq 2500 by Bionova Biotech Co. Ltd. For data analysis, the raw reads were mapped to the mm10 genome using bowtie (34 (link)), and then MACS software was used for peak calling. The motifs were called from significantly enriched peaks using the MEME suite (35 (link)). An in-house script was used to calculate the normalized and input-subtracted depth. The files were visualized in the UCSC Genome Browser. The microarray and ChIP-Seq data sets were deposited in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE133035).