Chip kit

The assay was conducted using a commercial ChIP kit from Active &Motif (cat#: 53040). Briefly, cells were seeded onto 15-cm plates and allowed to grow to 70%–80% confluence. Cells were fixed and collected, and the nuclear pellet was resuspended in ChIP Buffer, subjected to sonication and incubated overnight with 5-μg antibody, followed by incubation with protein G agarose beads for 3 h at 4 °C. DNA–protein complexes were eluted and de-cross-linked through a series of washes. Purified DNA was resuspended in TE buffer and analyzed with PCR using the following primers: E-cadherin-ChIP-F, 5′-ACTCCAGGCTAGAGGGTCACC-3′; E-cadherin-ChIP-R, 5′-CCGCAAGCTCACAGGTGCTTTGCAGTTCC-3′; TEL2-ChIP-E1-F, 5′-TGAATGTGCATTAGTTTATCAAGCC-3′; TEL2-ChIP-E1-R, 5′-CAATCTGCCTACCAGAAATTTATTC-3′; TEL2-ChIP-E2-F, 5′-CACAGTCACGGCTCACTGCAG-3′; TEL2-ChIP-E2-R, 5′-GAGTTGGACACCAGTCTGAACAAC-3′; TEL2-ChIP-E3-F, 5′-GGAGCGCTCAAGACAGAAAGC-3′; TEL2-ChIP-E3-R, 5′-AAAATAGGTTTGGAAATCTAGGTGG-3′; TEL2-ChIP-E4-F, 5′-AGGCAGTAGAGTGGTTAACACAAAC-3′; TEL2-ChIP-E4-R, 5′-TTTATGGAGTTCTCTGTGGATCATG-3′; GAPDH-ChIP-F, 5′-TTCTTGCCTTGCTCTTGCTACTC-3′; and GAPDH-ChIP-R, 5′-AGCCTGCCTGGTGATAATCTTTG-3′.

This procedure was performed as described by ChIP kit (Active&Motif, Catalog No.53040). Briefly, 15 cm plates for each cell line to be tested were seeded with cells that were allowed to grow to 70–80% confluence. To fix cells, 1/10 growth medium volume of Complete Cell Fixative Solution was added to the existing culture media for the cells. The fixation reaction was stopped by adding 1/20 media volume of Stop Solution to the existing culture media for the cells. The cells were collected by centrifugation. After centrifugation, the nuclear pellet was resuspended in ChIP Buffer. The cell lysate was subjected to sonication and then incubated with 5 μg of antibody overnight, followed by incubation with the protein G agarose beads for 3 hrs at 4°C. Bound DNA-protein complexes were eluted and cross-links were reversed after a series of washes. Purified DNA was resuspended in TE buffer for PCR. The primers for the SERPINE1 was as follows:
SERPINE1-ChIP-F: 5′- AGAGTCTGGACACGTG GGGAGTC-3′ SERPINE1-ChIP-R: 5′- CTCCATCAAAA CGTGGAAGTTTTC-3′

The ChIP assay was performed with the ChIP kit (Active Motif, Shanghai, China) according to the manufacturer’s protocol. The main steps were as follows: cell cross-linking and ultrasonic fragmentation of chromatin were performed. The anti-Sox2 antibody (CST), anti-RNA polymerase II (positive control), or normal mouse IgG (negative control) were added to the chromatin solution, respectively. The solution was incubated overnight at 4 °C. The protein and DNA were then de-crosslinked. The DNA was purified and enriched. Primers were designed and synthesized according to the predicted binding sites in the promoter region. qRT-PCR was performed with the primers.

Using a ChIP kit (Active Motif, Carlsbad, CA, USA), about 10⁷ cells were cross-linked and lysed. Chromatin was sheared to 300 to 700 bp fragments. Sheared chromatin DNA mixture (normalized inputs) was incubated with 4 μg FoxM1 antibody (#Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Negative control IgG and positive control H3 antibody were added at 10 μl (4 μg) per ChIP reaction. Real-time PCR were amplified using PTTG1 promoter primer (targeting −391 to −385 bp region), PTTG1 exon2 primer (as negative control) and VEGF promoter primer (as positive control).

For ChIP experiments, SQ20B cells were plated 24 h before placing into hypoxia chamber or standard incubator. Six 10-cm plates at 70% confluency were grown in normoxic or hypoxic (0.5% O₂) conditions for 16 h, at which time cells were crosslinked for 10 m at room temperature using 1% formaldehyde in minimum essential medium. Crosslinking was stopped and cells were washed and lysed according to manufacturer’s protocol (Active Motif ChIP kit #53035). Shearing conditions were carried out for six 20-s pulses at 25% power, with 30 s rest in between pulses on ice. Immunoprecipitations were carried out according to protocol, with 40 ug chromatin and 3 ug antibody for RNAPII (Active Motif #39097, mouse IgG #53010) and 60 ug chromatin with 3 ug (rabbit IgG Santa Cruz #sc-2027X) or 30 ug P-Ser2 RNAPII (Abcam #5095). For the resulting qPCR reactions, primers specific to EIF2B5 intron 10 and intron 12 were used (S1 Table), and control primers were purchased from Active Motif (GAPDH-2 for RNAPII control #71006, GAPDH-1 for P-Ser2 RNAPII control #71004, and Negative-1 for a 78-bp intergenic region of chromosome 12 as a negative control #71001).