Ab109228

The SimpleChIP Plus Enzyme Chromatin IP Kit (#9005, CST) was used for chromatin immunoprecipitation (ChIP) assays. We incubated for 20 min at room temperature for cross‐linking of the liver tissue mixture, made from mix of finely minced human liver tissue (10 mg) and 42.5 μL of 37% formaldehyde. We homogenise suspended tissue, washed twice with ice‐cold phosphate‐buffered saline (PBS) using a B‐type Dounce homogeniser, suspended by centrifugation after adding glycine to stop the cross‐linking reaction. Then, resuspended in Kit Buffer A and incubated with micrococcal nuclease for 20 min at 37°C. We disrupted nuclei by sonication, removed debris by centrifugation and treated clarified nuclear extracts with TET2 antibody (ab94580, Abcam, 1:50) or YY1 antibody (ab109228, Abcam, 1:100). Immunoprecipitation with protein G magnetic beads was performed after incubation at 4°C overnight. ChIP‐enriched DNA was analysed by qPCR using specific primers, as described in Table S3.

Cells were seeded on cell climbing sheets, fixed with 4% paraformaldehyde. After blocking with QuickBlock™ Blocking Buffer (P0260, Beyotime), cells were given primary antibodies to incubate, including RCAN1 (1:200, D6694, Sigma-Aldrich), YY1 (1:200, ab109228, Abcam), and NFATc1 (1:100, 66963-1-Ig, Proteintech). On the second day, Alexa Fluor 488 goat anti-mouse IgG (H + L) or Alexa Fluor 594 goat anti-rabbit IgG (H + L) secondary antibodies were employed to incubate cells, followed by nuclear staining (0100-20, SouthernBiotech) for 15 min. The fluorescence signals were analyzed by the thunder imager fast high-resolution inverted fluorescence imaging system (THUNDER DMi8, Leica, German).

Total cellular protein samples were separated with 10% SDS PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies against E-cadherin (ab40772), N-cadherin (ab76057), Vimentin (ab8978, Abcam), IGF2BP1 (ab82968, Abcam), YY1 (ab109228, Abcam), CDK4 (ab199728, Abcam), cyclin D1 (ab16663, Abcam), CDK2 (ab32147, Abcam) and GAPDH (ab9485, Abcam), along with HRP-conjugated secondary antibody were obtained and employed. GAPDH served as internal control.

A Simple ChIP Enzymatic Chromatin IP Kit (#9002, Cell Signaling Technology, Danvers, USA) was used to evaluate the accumulation of YY1, EP300, H3K9ac, and H3K27ac in RBM14 promoter according to the manufacturer’s instructions. Briefly, after being crosslinked with EBM-2 containing 1% formaldehyde, the crosslinked cells were collected in a lysis buffer containing 1% PMSF. Chromatin was digested by micrococcal nuclease, and 2% of aliquots of lysate were used as input control. Lysates were incubated with 3 μg primary antibody or normal rabbit IgG, followed by immunoprecipitation with protein G agarose beads and incubation at 4 °C overnight with gentle shaking. DNA crosslink was reversed by the addition of 5 mol/L NaCl and Proteinase K at 65 °C for 2 h. Immunoprecipitated DNA was purified and amplified by PCR using specific primers. The IgG (ab172730, Abcam), anti-EP300 (ab275378, Abcam), anti-YY1 (ab109228, Abcam), anti-H3K9ac antibody (ab32129, Abcam), and anti-H3K27ac antibody (ab4729, Abcam) were used. Immunoprecipitated DNAs were analyzed by qPCR. Primer sequences targeting RBM14 promoter (−91~+7; ch11: 66616629-66616727, named RBM14-promoter) were as follows: sense, 5′-CATTCCTGAGGAGGACTGCC-3′ and anti-sense, 5′-TCTTCATTTTGTCGCCGCAG-3′.

The details of western blot assays were performed as described in our previous published research [10 (link)]. Fresh tumor tissues and normal tissues treated with liquid nitrogen were crushed and then lysed in RIPA lysis buffer supplemented with proteinase inhibitor cocktail (Roche, number: 5892970001). In brief, lysed proteins were isolated by SDS-PAGE, transferred to PVDF membranes, and incubated with the following primary antibodies: anti-GAPDH antibody (1 : 3000 dilution, rabbit, Abcam, ab9485) andanti-YY1 antibody (1 : 500 dilution, rabbit, Abcam, ab109228). Following incubation with the appropriate HRP-conjugated secondary antibodies, the bands were visualized using Pierce™ ECL western blotting substrate (Thermo Scientific, number: 32106, USA). The signal intensity of the protein was further determined by ImageJ software (Madison, WI, USA).

We performed co‐immunoprecipitation (Co‐IP) using the Thermo Scientific Pierce Co‐IP kit. First, immobilised TET2 antibody (ab94580, Abcam, 1:50) with AminoLink Plus conjugated resin for 2 h, rinsed the resin and incubated it with tissue lysates overnight. Then, re‐washed the resin and eluted the protein using elution buffer using a primary YY1 antibody (ab109228, Abcam, 1:1000) and 800‐CW goat anti‐rabbit immunoglobulin G (IgG) as a secondary antibody (1:5000; LI‐COR Biosciences Inc.) Finally, membranes were analysed using the Odyssey infrared scanner (LI‐COR Biosciences Inc.).