Anti setd8

ChIP experiments were performed using a Simple ChIP plus sonication chromatin IP kit (Cell Signalling Technology, MA). Briefly, HGECs (1 × 10⁷) were fixed with 1% formaldehyde for 10 min at room temperature to cross-link the DNA and the proteins, then terminated by adding glycine. The chromatin was sheared by using a Microson XL ultrasonic cell disruptor XL (Misonix). Ten microliters of sonicated solution was spared as the input. The remaining was incubated with anti-MZF1 (Sabbiotech, USA, #33737), anti-SETD8 (Pro-teinTech, Wuhan, China, 14063-1-AP), anti-H4K20me1 antibody (Abcam, Cambridge, UK, ab9051) and anti-Histone H3 (Abcam, Cambridge, UK, ab1791) or negative control IgG, respectively, at 4 °C overnight. After purification, the enriched DNA sequences were detected by PCR and agarose gel electrophoresis on a 2% agarose TAE gel. The WNT5A oligonucleotide primer sequences were as follows: prime 1 (P1): forward, 5′-TTTTGCGACACCTGTGCTTG-3′, and reverse 5′-TGCCTGTTCTGTCCACTCA-3′; prime 2 (P2): forward, 5′-TAGTACAAGAGAATGGACTA-3′, and reverse 5′-CACCAGCCCAGCTCACCTCA-3′; prime 3 (P3): forward, 5′-CCAGATTGTATATTTGATGC-3′, and reverse, 5′-AGATAGTAAGTGGTGGAGCC-3′; primer 4 (P4): forward, 5′-TACCAGGCTGACACACTGCT-3′, and reverse 5′-GAATTACTGATCTGTAATAA-3′; prime 5 (P5): forward, 5′-ACTGTCCTCAGAGCTCTTGA-3′, and reverse 5′-AGCCACCACATGTGACCTTC-3′.

Masson staining was preformed to evaluate the severity of fibrosis in tissues according to the instructions (Solarbio, Beijing, China). Standard immunohistochemistry (IHC) procedures were performed by using anti-WNT5A (Sabbiotech, USA, #49719), anti-p-p65 (ProteinTech, Wuhan, China, 14063–1-AP), anti-MZF1 (Sabbiotech, USA, #33737), and anti-SETD8 (ProteinTech, Wuhan, China, 14063-1-AP) antibodies.

The clinical tissue samples used in this study were clinical PDAC tissue samples from 80 patients confirmed by surgery and pathology and approved by the ethics committee of the Affiliated Tumour Hospital of Fudan University (FUSCC). The immunohistochemical staining of paraffin-embedded tissues adopts a two-step scheme. First, The antigen was extracted by dewaxing hydration and antigen retrieval, and then the slide was incubated with the following primary antibodies: anti-SETD8 (Proteintech) and anti-RRAD (Abcam). HRP binds affinity-purified sheep anti rabbit IgG (Proteintech) as a secondary antibody. Three different views were randomly selected under the microscope, and each slide was scored. According to the total area and intensity of staining, the protein expression level score was (1), < 5% of the total cells; (2), 5–25%; (3), 25–50%; (4), 50–75 and > 75%: (5). The final score was the average of the three views and was classified as follows: low (1 ≤ score < 3) and high (3 ≤ score ≥ 5).