Bs 2006r

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.

Cells treated as described earlier were washed three times with PBS and lysed in RIPA buffer with a 1% protease inhibitor. Cell lysates were centrifuged, and the protein concentration was measured using a BCA assay kit. Equal protein aliquots (10 μg) were fractionated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 3% bovine serum albumin in TBST and incubated with primary antibodies of Bax (bs-0127R, Bioss, Beijing, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), PCNA (bs-2006R, Bioss, Beijing, China), LC-3 (12741S, CST, Boston, United States), Atg-5 (10181-2-AP, Proteintech, Wuhan, China), Beclin-1 (bs-1353R, Bioss, Beijing, China), NF-κB (10745-1-AP, Proteintech, Wuhan, China), p-NF-κB (bs-0982R, Bioss, Beijing, China), STING (19851-1-AP, Proteintech, Wuhan, China), cGAS (ab252416, Abcam, Cambridge, United Kingdom), iNOS (ab15323, Abcam, Cambridge, United Kingdom), GBP5 (13220-1-AP, Proteintech, Wuhan, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), and GAPDH (PMK053C, BioPM, Wuhan, China) overnight at 4°C, and then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were developed using an ECL, and the films were exposed using a bio-imaging system (170-8265, Bio-Rad).

By adding 1 mL of RIPA buffer and 10 µL of benzylmethylsulfonyl fluoride (Solarbio, Beijing, China), total protein was extracted. The quantification of all proteins was performed using an enhanced BCA protein assay kit (Thermo Fisher Scientific, Niederelbert, Germany). Following denaturation, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% skim milk at 24 °C for 2 h. The membrane was then incubated with polyclonal antibodies against SPC (1:1000; AP53886PU-N; OriGene, Maryland, USA), HIF-1α (1:1000; AF1009; Affinity, Changzhou, China), EGFR (1:300; bs-10007R; Bioss, Beijing, China), EGF (1:300; bs2010R; Bioss Beijing China), PCNA (1:300; bs-2006R; Bioss Beijing China), Bax (1:300; bs-20386R; Bioss Beijing China), and β-actin (1:2000, Bioss, Beijing, China). After washing with PBS at 24 °C for 90 min, the membrane was further incubated with IgG antibody (1:3000, bs-0295G-HRP, Bioss, Beijing, China). Finally, an ECL luminescent solution was used for color development and the results were observed. β-actin served as the loading control. Grayscale values of protein bands were analyzed using ImageJ software to determine relative expression levels.