Anti pcna antibody

Prior to IHC, the tumor tissues were immobilized, dried, and implanted in paraffin, the tumor tissue specimens were stripped into 3 μm samples to store. In IHC process, specimens were first deparaffinized and rehydrated using xylene and alcohols, respectively. After restoring the antigen with the sodium citrate reagent, the tissues were washed thrice by PBS ensued by blocking with H₂O₂ and BSA (Thermo Scientific, USA). Then, incubation of the samples was done with the primary anti‐FUT11 antibody (1:100; Proteintech, China), anti‐COL6A3 antibody (1:100; Abcam, USA), anti‐Ki67 antibody (1:100; Proteintech, China) and anti‐PCNA antibody (1:100; Proteintech, China) for 24 h at 4 °C. On the second day, staining of the sections was done with HRP‐conjugated goat anti-rabbit secondary antibodies (ABclonal, Wuhan, China) for 2 h. After washing thrice by PBS, the tissues were subjected to incubation with the DAB (Beyotime Biotechnology, Suzhou, China) and hematoxylin, immune signals were detected by an orthophoto microscope.

Tumor tissues were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 5 µm using routine methods. For immunohistochemical staining, sections were incubated with the anti-PCNA antibody (Proteintech) overnight at 4°C. PCNA staining was visualized using the ImmPress HRP Reagent kit (Vector Laboratories, Burlingame, CA, USA) with NovaRed as a substrate (Vector Laboratories). Hematoxylin was used as a counterstain for the cell nucleus. For the quantification analysis, we counted the number of total cells and PCNA-positive cells per 400× microscope field with 3random fields per mouse. The data are expressed as the percentage of PCNA positive cells, which was calculated as [(positive cell number) / (total cell number)] ×100.

Dihydrotanshinone I (purity ≥ 98%) was obtained from Chengdu MUST Biological Technology Co., Ltd. (Chengdu, China). Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-DDDDK tag antibody (cat#20543-1-AP), anti-PCNA antibody (cat#10205-2-AP), anti-importin Α5 (cat#18137-1-AP), Anti-PKM2 antibody (cat#60268-1-Ig), anti-cardiac troponin T antibody (cat#15513-1-AP), anti-GAPDH antibody (cat#60004-1-Ig), goat anti-rabbit IgG (cat#SA00001-2), anti-GLRX antibody (cat#15804-1-AP) and goat anti-mouse IgG (cat#SA00001-1) were obtained from Proteintech (Wuhan, China). Alexa Fluor 594 (cat#ab150116), alexa-fluor 488 (cat#ab150077), anti-glutathione antibody (cat#ab19534), mito-TEMPOL (cat#ab144644) and cycloheximide (cat#ab120093) were obtained from Abcam (Cambridge, UK). Anti-FLAG magnetic beads (cat#M8823), wheat germ agglutinin (cat#L9640), diamide (cat#D3648), isoprenaline (cat#I5627) and N-acetylcysteine (cat#A7250) were obtained from Sigma–Aldrich (St. Louis, MO, USA).