Dab color reagent kit

The NPC tissue specimens (n=36) were collected from the Nanfang Hospital of Southern Medical University (Guangzhou, China). These tissue specimens were from biopsy samples of NPC patients (pathologically confirmed) in 2021. The TNM classification was performed according to the 8th edition of the UICC/AJCC staging criteria. The clinical characteristics of these 36 patients with NPC are shown in Supplementary Table 3. This study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University. Formalin-fixed, and paraffin-embedded tissues were sectioned at 4 mm thickness, then harvested and fixed in 4% paraformaldehyde overnight at 4°C. The antigen blocking was performed using 10% goat serum (SP-9000, Zsbio, China). The sections were probed overnight at 4°C with primary anti-TGF-β1 (PA5-40772, Invitrogen, USA), anti-c-Jun (AF6089, Affinity, USA) and anti-STT3A (sc-100796, Santa Cruz, USA) antibodies. DAB color reagent kit (ZLI-9018, Zsbio, China) was used for staining. The numbers of positive cells were measured by two independent pathologists through five randomly selected fields at ×400 magnification.