Anti e2f1

HCT-116 cells overexpressing RFWD3 were cross-linked with formaldehyde, lysed in SDS buffer, and mechanically sheared by sonication to fragment DNA. Protein–DNA complexes were precipitated using 2 μg control normal mouse IgG (Sigma, Cat. No. I5381), 2 μg Histone H3 (D2B12) XP^® Rabbit mAb (CST, Cat. No.4620) and 4 μg anti-E2F1 (Proteintech, Cat. No. 66515-1-Ig) antibody. After that, the eluted DNA fragment was detected with the primers specific for BIRC5 promoter and SYBR premix (Vazyme).

Western blot assays were conducted as previously described.8 The associated primary immunoblotting antibodies were as follows: anti‐GAPDH, anti–E‐cadherin, anti–N‐cadherin, anti‐Vimentin, anti‐CDK6, anti‐CCND1, anti‐CDK4, anti‐NOP14, anti‐MMP9, anti‐MMP2, anti‐E2F1, anti‐MET, anti‐Parp‐1, (Proteintech Group), anti‐Caspase 3, anti‐DNMT3B (Cell Signaling Technology) and anti‐SP1 (Santa Cruz Biotechnology).

6-well plates were used for culturing HCFs. HCFs were collected after treatments. BCA protein assay kit was used for determining the concentrations of cell lysates. 1 mg of total proteins were used for immunoprecipitation. The samples were incubated in rabbit-controlled Ig-G antibody and anti-E2F1 (1:100, Proteintech, Wuhan, China) antibody under condition of 4°C overnight. Then the samples were suspended with 25 μL of protein A/G PLUS-Agarose. After 2 h of incubation and washing, the samples were western-blotted, and anti-CCNE2 (1:1000, Abclonal, Wuhan, China) antibody was incubated to evaluate the conjunction amount of E2F1 and CCNE2.

ChIP assays were performed as previously described [19 (link)]. In brief, the ChIP assay was conducted according to the protocol of the ChIP assay kit (CST). C4-2 and PC3 cells were cultured in 10-cm dishes. Then, 243 µL of 37% formaldehyde was added to one plate to a final concentration of 1% (the culture medium had a total of 9 mL). After incubating at 37°C for 10 min, the chromatin was cross-linked, followed by rinsing twice with 2 mL of precooled PBS including protease inhibitors. The cells were then lysed in ChIP lysis buffer and sonicated to entirely disrupt the nuclei. The ChIP buffer was used to dilute the digested, cross-linked chromatin. A 10 µL sample was extracted from the diluted chromatin as a 2% input group. Then, 500 µL of the remaining diluted chromatin and corresponding antibodies (anti-E2F1, ProteinTech, catalog no. 66515-1-Ig, or anti-IgG, Beyotime Institute of Biotechnology, catalog no. A7001) were incubated overnight at 4°C with rotation. Following elution of chromatin, reversion of cross-links, and DNA depuration, qRT-PCR was carried out to identify the potential E2F1 binding sites on the RACGAP1 promoter region.

Western blot assay were conducted according to previous description^{32 (link)}. The primary antibodies used include the following: anti-MCPIPI (#GTX110807, GeneTex, 1:1500), anti-cyclin D1 (#A19038, ABclonal, 1:1000); anti-RB (#25628–1-AP, Proteintech, 1:1000), anti-pRB(Ser780) (#9307, Cell Signaling Technology, 1:1000), anti-E2F1 (#12171–1-AP, Proteintech, 1:1000), anti-Flag-Tag (#T0003, Affinity Biosciences, 1:5000), anti-GAPDH (#60004–1-Ig, Proteintech, 1:5000), anti-β-actin (#60008-1-Ig Proteintech, 1:1000). The secondary antibodies used include HRP-conjugated goat anti-mouse antibody (#SA00001-1, Proteintech, 1:10,000) and HRP-conjugated goat anti-rabbit antibody (#SA00001-2, Proteintech, 1:10,000). The bands were detected with ECL reagent (Advansta, USA). Image J software was used to calculate the protein expression levels.

Western blot analysis was conducted as previously described.^{14 (link)} The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Proteins were extracted using lysis buffer, separated via SDS‒PAGE, and transferred onto PVDF membranes; the membranes were then blocked with 5% nonfat milk in TBS/Tween 20 and then incubated with the indicated primary and secondary antibodies. The antibodies used were as follows: anti-NSUN2 (1:500 dilution; Proteintech), anti-YBX1 (1:2000 dilution; Proteintech), anti-E2F1 (1:1000 dilution; Proteintech), anti-ALYREF (1:500 dilution; Proteintech), anti-Lin28B (1:500 dilution; Proteintech), anti-GAPDH (1:20000 dilution; Proteintech), anti-Tubulin (1:1000 dilution; Proteintech) and anti-Flag (1:2000 dilution; MBL).