Proliferating cell nuclear antigen (pcna)

The paraffin-embedded sections of the neoplastic tissue were dewaxed, rehydrated, and antigen-repaired with sodium citrate for 30 min. Then, they were incubated with 3% hydrogen peroxide at room temperature for 20 min. Next, the sections were blocked with 3% BSA for 1 h and labeled with a primary antibody at 4 °C overnight. The following primary antibodies were used: CDK4 (11026-1-AP, Proteintech, Wuhan, China), CydlinD1 (26939-1-AP, Proteintech, Wuhan, China), CDC25A (55031-1-AP, Proteintech, Wuhan, China), Bax (bs-0127R, Bioss, Beijing, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), PCNA (bs-2006R, Bioss, Beijing, China), Ki67 (bs-23103R, Bioss, Beijing, China), B-Myb (ab12296, Abcam, Cambridge, United Kingdom), γ-H2A.X (bs-3185R. Bioss, Beijing, China), and p53 (bs-2090R, Bioss, Beijing, China). The sections were stained with iNOS, GBP5, and CD11b to analyze the phenotype of macrophages. The samples were subsequently stained with a secondary antibody (PV-9000, ZSGB-BIO, Beijing, China) at 37 °C for 1 h. Diaminobenzidine (DAB, ZL-9018, ZSGB-BIO, Beijing, China) was used for staining at room temperature for 1–3 min. The nuclei were stained with hematoxylin or DAPI. Finally, the paraffin-embedded sections were observed with an orthogonal Olympus microscope or a confocal microscope.

The expression levels of PCNA (1:200, rabbit IgG, Bioss, Beijing, China), CD68 (1:200, rabbit IgG, Bioss, China), and PCNA (1:100, Immunoway Biotechnology, Plano, TX, USA), were detected by immunofluorescent, immunohistochemical, and EdU analysis as previously described [36 (link),37 (link)].

Cells were rinsed twice in ice-cold PBS. The RIPA buffer (Solarbio), protease inhibitor mixture (Solarbio), and protein phosphatase inhibitor (Solarbio) were subsequently added to the extract protein. Supernatants were used to quantify the protein concentration via the Bradford protein kit (Tiandz, Beijing, China). Proteins were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 40 min and 120 V for 50 min, and subsequently electrotransferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for 80 min. Membranes were incubated with primary antibody P21 (Proteintech, Wuhan, China), P53 (Bioss, Beijing, China), PCNA (Bioss), p-Cyclin B1 (Bioss), Claudin1 (Bioss), Occludin (Bioss), ZO-1 (Bioss), Cdc25c (CST, Boston, MA, USA), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. After incubation with corresponding secondary antibodies, the protein band densities were quantified using Bio-Rad ChemiDoc™ XRS and Image Lab™ Software.

CNE2 cells with different concentrations of luteolin (0 μM, 20 μM, and 40 μM) and CIS (10 μM) were incubated for 36 h, total protein was extracted and quantified, and 50 μg of protein was used for western blot analysis. The appropriate concentration of separating gel and stacking gel was chosen depending upon the molecular weight of the target protein. The samples were then loaded, onto the SDS-PAGE gel, electrophoresed and the resolved proteins were transferred to a transfer membrane (PVDF), and blocked. Diluted β-actin (Cell Signaling Technology, CST, MA, USA), p-ERK1/2 (CST), ERK1/2 (CST), AKT (CST), PI3K (CST), PCNA (Bioss, Woburn, MA, USA), and XIAP (CST) at a dilution ratio of 1 : 1,000 were incubated with the PVDF membrane overnight at 4°C, then wash three times with TBST, for 10 min each. Then, the corresponding diluted goat anti-mouse or goat anti-rabbit secondary antibody was added, and incubated with the PVDF membrane at room temperature for 2 h, washed with TBST three times for 10 min and then scanned on an ODYSSEY CLx Infrared Imager (LICOR, Lincoln, NE, USA) to detect the signal intensities of the immunoreactive bands. Using β-actin as an internal reference, the comparative protein expression levels could be calculated, the experiment was performed in triplicate.