Nb500 170

For hematoxylin and eosin (H&E) staining, tissues were fixed in formalin, paraffin-embedded, sectioned (5 μm), deparaffinized and stained with H&E staining kit (Solarbio, G1120-3). For IHC staining, sections were deparaffinized and treated with 3% H₂O₂ in water for 10 min. Antigen retrieval procedure was conducted to sections with 10 mmol/L citrate buffer (pH 6.0) for 10 min. Next, tissue sections were incubated with the Biocare blocking reagent for 10 min, followed by an overnight incubation at 4 °C with anti-NAT10 (Santa Cruz, sc-271770 1:200), anti-RNPS1 (Proteintech, 10555-1-AP, 1:200), anti-Ki67 (Novus, NB500-170, 1:200), anti-PCK (pan-Cytokeratin, Santa Cruz, sc-8018, 1:200). Slides were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature, treated with 3,3′‐diami‐nobenzidine and counterstained with hematoxylin. For analysis, the staining intensity was scored and the percentage of positive stained areas of tumor cells per the whole tumor area were calculated.

All specimens collected were routinely embedded in paraffin, and the tissue slides were deparaffinized and rehydrated. H&E staining was performed with an H&E staining kit (Solarbio, G1120-100). For IHC staining, after incubation with 3% H₂O₂, antigen retrieval, and blocking, sections were incubated with primary anti-PCIF1 (Novus, NBP2-13740), anti-CTBP2 (Proteintech, 10346), anti-TET2 (Abcam, ab94580), anti-Ki67 (Novus, NB500-170), and anti-PCK (pan-cytokeratin; Santa Cruz, sc-8018) overnight at 4°C. After washing, secondary antibodies and streptavidin-biotin complex (SABC) were applied with an SABC-POD kit (BOSTER, SA1022). Then the slides were stained with a 3,3′-diaminobenzidine (DAB) kit (BOSTER) and counterstained with hematoxylin. For the IHC analysis, the IHC score was calculated by multiplying the proportion of positively stained cells at each intensity level by their respective intensity score (intensity scores ranging from 0 to 3). The final score ranges from 0 to 300. For Ki67, the percentage of positively stained cells was calculated. The anti-PCK antibody (Santa Cruz Biotechnology, sc-8018) was used for IHC staining of metastatic cells in cervical lymph nodes.

For immunological analyses, 3–5-μm microtome sections were deparaffinized for 30 min and rehydrated with gradient alcohol. Then, the endogenous peroxidase activity of the sections was quenched with 3% H₂O₂, and heat-induced epitope retrieval was performed. The sections were incubated with primary antibodies targeting the following proteins at 4 °C overnight: SOX9 (1:200; Millipore, AB5535), Ki67 (1:500; Novus, NB500170), CD45 (1:200; Santa Cruz, sc-53665), α-SMA (1:200; CST,19245S), PDPN (1:200; abcam, ab256559), CC10 (1:200; Santa Cruz, sc-365992), Caspase-3 (1:200; CST, 9661S), SOX2 (1:300; abcam, ab97959), Oct3/4 (1:300; BD, BD611203), Klf4 (1:300; CST, 12173S) and cMyc (1:300; CST, 5605S). The specimens were washed with phosphate-buffered saline (PBS) three times prior to incubation with secondary antibodies targeting the primary antibodies for 30 min at 37 °C. For enzymatic assays, a horseradish peroxidase (HRP) conjugate was used for detection. The IHC score was calculated by multiplying the percentage of positive cells by the intensity. The intensity was scored as 0 (negative), 1+ (weak staining), 2+ (moderate staining) or 3+ (strong staining), while the frequency was scored according to the proportion of positive cells.