Suv39h1

Immunoblotting was conducted as described previously [24 (link)]. Colon tissues from mice were dissected the next day after completing the WAS procedure. The epithelial layers were gently scraped off, and proteins were extracted. In separate studies, human LS174T cells were collected and processed with the same method. Proteins were separated and blotted with the following primary antibodies: Piezo1 (1:500, 15939-1-AP, Proteintech), H3K9me3 (1:500, 13969, Cell Signaling Technology), T-H3 (1:500, GTX122148, GeneTex), SUV39h1 (1:500, PA5-29470, Thermo-Fisher), Histone Deacetylase 3 (Hdac3) (1:500, GTX1096679, GeneTex), and GAPDH (1:1000; Proteintech).
For immunofluorescence staining, paraffin sections were stained as described in previous research [25 (link)]. Primary antibodies used for incubation were as follows: Piezo1 (1:200, 15939-1-AP, Proteintech), H3K9me3 (1:500, 13969, Cell Signaling Technology), AGR2 (1:200, AF6068, R&D Systems), and Mucin2 (1:200, GTX100664, GeneTex). DAPI (D9542, Sigma, Millipore, St. Louis) was used for nucleic acid staining.

Whole-cell lysates were prepared as described in [21 (link)]. A histone-containing fraction was obtained by acidic extraction following the protocol available at (https://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot). Protein concentration was determined using a protein assay (Bio-Rad, Hercules, CA, USA). Samples containing 80 µg protein were precipitated first by adding a 1:10 volume of 100% trichloroacetic acid for 30 min on ice and, after a 10 min centrifugation at 12,000× g, cold acetone for 16 h at −20 °C. Samples were subjected to SDS-PAGE and immunoblotting. Blots were examined with antibodies recognizing SUV39H1 (rabbit polyclonal, Thermo Fisher Scientific, 1:1000), histone H3 (rabbit polyclonal, 1:1000, Merck Millipore, Burlington, MA, USA), H3K9me3 (rabbit polyclonal, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA), and β-actin (HRP-conjugated mouse monoclonal, Sigma Aldrich, 1:20,000). In the case of dot blots, cells (1 × 10⁵) from individual cell clones were collected and lysed in 10 µl of lysis buffer (Promega—Cell Signaling technology, Danvers, MA, USA) for 30 min on ice with occasional vortexing, and the resulting lysates were directly transferred on nitrocellulose and processed as in a Western blot.

Small interfering RNA (siRNA) mediated gene knockdown was performed using Hyperfect transfection reagent (Qiagen, Hilden, Germany) following the manufacturer's instructions. A total of 20 pM siRNA were transfected per 2 × 10⁴ cells. Cells were grown for 72 h before IR. SMARTpool siRNA oligonucleotides from Dharmacon were used for Control, HP1α, HP1β, HP1γ, CtIP, EXO1 and BLM. SUV39H1 (5′-ACCUCUUUGACCUGGACUAT-3′) and SUV39H2 (5′-GAAGCUACCUUUGGUUGUUTT-3′) siRNA oligonucleotides were obtained from Thermo Scientific. SETDB1A (5′-GGAACUGGAGAAGAUGGAUUGUGUA-3′) and SETDB1B (5′-CCGTGAAGCTATGGCTGCCTTAAGA-3′) were from Dharmacon. BRCA1 (5′-GGAACCUGUCUCCACAAAG-3′) siRNAs were synthesized by Invitrogene (UK). Unless otherwise stated, combined HP1α/β siRNA oligonucleotides were used. Combined SETDB1A/B and combined SUV39H1/H2 were used in all experiments.
The pCBASceI (from Dr M Jasin) plasmid was transfected using GeneJuice transfection reagent (Novagen, Germany) per 2.5 × 105 cells at 24 h post siRNA transfection.