Ultrasonic cell disruptor

ChIP assays were performed with the Agarose ChIP kit (Pierce, USA) according to the manufacturer’s instructions. After treatment with or without 10 μM As₄S₄ for 12 hours, HCT116 cells were subjected to cross-linking with 1% formaldehyde. Glycine solution was then added and DNA was sonicated to an average size of 100–300 base pair by an ultrasonic cell disruptor (Diagenode, Belgium). Co-immunoprecipitation was performed overnight at 4˚C with ChIP-Validated Monoclonal Anti-p53 (Sigma-Aldrich) or anti-NFATc3 by incubation at 4 °C for overnight on a rocking platform. Precipitates were washed and samples were extracted twice with elution buffer, heated at 65 °C to reverse crosslinks and DNA fragments were purified with phenol/chloroform and suspended in normal saline and used for RT-PCR. Please see Supplementary Information for the primer sequences.

ChIP was performed with MagnaChIP HiSens Chromatin IP Kit (17–10461, Merck Millipore) following the manufacturer’s protocol. In brief, cells were cultured under hypoxia for 32 h and then cross-linked with formaldehyde at 37 °C for 10 min, quenched with glycine, and then sonicated to generate 300–600 bp DNA fragments using an Ultrasonic Cell Disruptor (Diagenode, Liège, Belgium). The antibodies against HIF1α (610959, BD), PKM2 (4053 S, CST), biotin-ab (033700, Invitrogen), p300 (ab54984, Abcam), H3K9ac (ab4441, Abcam), were used to for immunoprecipitation. The binding of the HIFAL promoter to HIF1α, PKM2, biotin-ab, or IgG was quantified using quantitative PCR with primers. Chip primers sequences were listed in Supplementary Table 1.

A ChIP assay was performed using a Magna ChIP HiSens chromatin IP kit (Merck Millipore) according to the manufacturer’s instructions. Caki-1 cells pretreated with TGF-β1 (10 ng/mL) for 2 h were fixed with 1% formaldehyde for 10 min at 37°C. The fixed cells were quenched with glycine and collected for sonication in an ultrasonic cell disruptor (Diagenode, Belgium) to generate DNA fragments ranging from 300 to 600 bp. IPs were performed with an antibody against Smad3 (Cell Signaling Technology, Boston, MA, USA). The binding of the promoter region of lnc-TSI to immunoglobulin G (IgG) or Smad3 was quantified using real-time PCR. Precipitated DNA fragments were detected with specific primers as follows: sense, 5′-ACAGACGGGAGTCGAGTGTA-3′, antisense, 5′-CAGACATGGGCAATCCCACT-3′.