Ab50917

The tumors from the nude mice were fixed in 10% paraformaldehyde at 4°C for 12 h, and then dehydrated in different concentrations of ethanol, permeabilized using xylene and embedded in paraffin. The paraffin-embedded tumor tissues were then sliced (0.5 µm), rehydrated, and were stained with HE at 4°C for 10 min and sealed with neutral gum. For IHC assessment of E2F3 and Ki-67 in colorectal tumor tissues from the nude mice, the DAKO Envision system (Dako; Agilent Technologies, Inc.) was used. Briefly, after paraffin-embedded sections of tumor tissues were heated at 60°C, the sections were incubated with primary antibodies against E2F3 (1:200; cat. no. ab50917; Abcam) and Ki-67 (1:1,000; cat. no. ab279653; Abcam) overnight at 4°C. Then, the sections were incubated with biotin-labeled secondary antibodies (1:1,000; cat. no. ab205718; Abcam) at 37°C for 20 min. Under a light microscope, IHC staining scores for E2F3 were obtained by multiplying the intensity (0, negative; 1, low; 2, medium; and 3, high) with the extent of staining (0, 0%; 1, 0-10%; 2, 10-50%; 3, 50-75%; 4, >75%); the final scores were between 0-12. For evaluation of Ki67, the number of positive cells was calculated in three representative areas of high staining.

Cells were transfected for 48 h and lysed using RIPA buffer (catalog# 9806S, CST, Danvers, MA, USA). The samples were centrifuged, and proteins in the supernatants were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 10% or 12% gels and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by incubation with skim milk(1:5000; Boster, Wuhan, China) and then incubated for 12 h at 4°C with primary antibodies against E2F3 (catalog# ab50917; Abcam, Cambridge, UK), PRDM1 (catalog# ab106766, Abcam), cyclin-dependent kinase 17 (CDK17, catalog# ab159068, Abcam), or β-actin (catalog# ab8227, Abcam). After washing, the membranes were incubated for 2 h at 37°C with anti-rabbit, anti-mouse, or anti-goat IgG secondary antibodies. Electronic chemical Laboratory (ECL) detection kit was used for the signal development (Merck Millipore, Darmstadt, Germany).

Total protein was isolated using RIPA buffer (Beyotime, Shanghai, China) and examined using a BCA protein assay kit (Tiangen, Beijing, China). Equal of proteins were subjected to sodium dodecyl sulfonate‐polyacrylamide gel (SDS‐PAGE; Solarbio) and transferred onto polyvinylidene difluoride membranes (Millipore). Next, the samples were blocked with 5% non‐fat milk for 1 hour at room temperature. Afterward, the samples were incubated with primary antibodies: E2F3 (ab50917; Abcam) or GAPDH (ab181602; Abcam) overnight at 4°C and relevant secondary antibody (ab205719; Abcam) for 2 hours at room temperature. Finally, enhanced chemiluminescence reagent (Vazyme, Nanjing, China) was utilized to visualize the proteins.