Ab40890

Cells treated with 8PN and/or EGFR TKI (afatinib or AZD9291) were lysed in NETN lysis buffer (150 mm NaCl, 1 mm EDTA pH 8.0, 20 mm Tris–HCl pH 8.0, 0.5% NP40) after 24 h. Immunoblotting was conducted as previously described [6 (link)], except that we used total of 10% sodium dodecyl sulfate–polyacrylamide gels for protein separation. After 1 h of blocking, primary antibodies were used against the following targets: CDCP1 (ab1377; Abcam, Cambridge, MA, USA), retinoblastoma protein (RB; #2947; Cell Signaling Technology, Danvers, MA, USA), phosphorylated RB (#8516; Cell Signaling Technology), cyclin E1 (ab33911; Abcam), cyclin E2 (ab40890; Abcam), EGFR (sc‐373746; Santa Cruz, Dallas, Texas, USA), phosphorylated EGFR (p‐EGFR; ab1377 and ab32894; Abcam), phosphorylated ERK (p‐ERK; #9101; Cell Signaling Technology), ERK (#9102; Cell Signaling Technology), and caspase 3 (#9661s; Cell Signaling Technology, or ab1899; Millipore, Billerica, MA, USA). GAPDH (10494‐1‐AP; ProteinTech, Rosemont, IL, USA), and EF1α (#05‐235; Millipore) were used as internal controls. Blotted proteins were detected using an enhanced chemiluminescence system (Millipore) with the BioSpectrum Imaging System (UVP, Upland, CA, USA).

MCF-7 and T47D cells were lysed in lysis buffer (#KGP250/KGP2100, Keygen Biotech, Nanjing, China) containing protease and phosphatase inhibitors following the manufacturer’s protocol. Proteins were subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a PVDF membrane, then incubated with CCNE2 (#ab40890, Abcam; dilution rates of 1:3000), CDCA5 (#67418-1-Ig, Proteintech; dilution rates of 1:1000), RAD51(#14961-1-AP, Proteintech; dilution rates of 1:1000), MCM10 (#12251-1-AP, Proteintech; dilution rates of 1:500), ERα(#ab108398, Abcam; dilution rates of 1:2000), PR(#ab206926, Abcam; dilution rates of 1:1000), Vinculin(#26520-1-AP, Proteintech; dilution rates of 1:2000) and GAPDH antibodies (#T0004, Affinity; dilution rates of 1:5000) at 4 °C overnight, respectively. The next day, the membranes were exposed to secondary antibodies (#S0101, #S0100, Lablead, Beijing, China; 1:5000 dilution) at room temperature for 1 h. Protein bands were measured using Syngene GeneGenius (SYNGENE, GeneGnome XRQ NPC, Cambridge, UK.).

Immunofluorescence and immunohistochemical staining was performed following the manufacturer’s instructions. Culture cells were fixed using paraformaldehyde, followed by permeabilized with 0.1% Triton X-100, and blocking using 10% Block Aid^TM (B10710, Invitrogen). The primary antibodies used in this study were B-cell lymphoma 2 (BCL2) (1:200, ab182858, Abcam, CA, USA), proliferating cell nuclear antigen (PCNA) (1:200, ab92552, Abcam, CA, USA), and cyclin E2 (CCNE2) (1:200, ab40890, Abcam, CA, USA). Subsequent to primary antibody incubation, the cells were washed and incubated with 2 µg/mL Donkey Anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-21207-1, Thermo Scientific, CA, USA) for immunofluorescence staining or for immunohistochemical staining. Following staining, nuclei were counterstained using diamidino-2-phenylindole (DAPI) and the cells were imaged using a laser scanning confocal microscope. Negative controls were stained without primary antibodies or control IgG.

Ethical guidelines were followed during all animal studies, as approved by the Ethics Committee of Wannan Medical College (No. 2021012). Ad-FLuc was provided by Prof. Tong-Chuan HE (University of Chicago). HCT116 and HCT116-siKCN cells were infected with Ad-Fluc to produce HCT116-FLuc and HCT116-siKCN-FLuc, respectively. 4–6-week-old BALB/c nude mice (male/female ratio 1:1) were subcutaneously injected with a total of 1.5 × 10⁷ cells per injection were collected and injected subcutaneously into the right dorsal flank of the mice in 200 mL of PBS (4 mice per group) for the tumorigenesis assay. The animals were intraperitoneally injected with luciferase (ab145164, Abcam) after 2 and 14 days of injection, and bioluminescence imaging was performed using the Berthold LB983 imaging system. INDIGO software (Berthold Technologies) was used to analyze the data from bioluminescence images. The mice were euthanized after 20 days of injection and the tumors were collected and analyzed after making paraffin sections. Immunofluorescence staining using anti-PCNA (1:400, ab92552, Abcam) and anti-CCNE2 (1:400, ab40890, Abcam) was conducted to detect and quantify positive cells in three randomly selected high-power fields of each section.

Total proteins were extracted from the cells, followed by quantification using the RIPA lysis buffer and a BCA detection kit (Beyotime, China), according to the manufacturer’s instructions. Equal amounts of protein were then separated using 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then blocked, followed by incubation with primary antibodies against BCL2 (1:1000, ab182858, Abcam), caspase3 (CASP3) (1:1000, 9662s, Cell Signaling Technology), PCNA (1:1000, ab92552, Abcam), CCNE2 (1:1000, ab40890, Abcam), and β-actin (1:1000, ab8226, Abcam) at 4°C for one night. Peroxidase—conjugated secondary antibodies were then used for incubation at room temperature. Chemiluminescence (Beyotime) was used for the detection of the bands, with protein bands quantified using Image J software (http://www.imagej.nih.gov/ij/v2.4.1).