Cy5659

Cells were lysed in the EBC250 lysis buffer (250 mM NaCl, 25 mM Tris pH 8.0, 0.5% Nonidet p-40, and supplemented with 50 mM NaF, 20 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/mL leupeptin). Western blotting analysis were performed as described⁵⁴ . Antibody specific for E2A (sc-133074) was purchased from Santa Cruz Biotechnology (Burlingame CA, USA). Antibodies specific for p63 (CY5659), p21 (CY5543), and GAPDH (AB0037) were purchased from Abways (Shanghai, China). Antibodies specific for CyclinE1 (3327) and CyclinD1 (2261) were purchased from Epitomics (Burlingame CA, USA). GAPDH was used as loading control.

Tissue microarray slides of human squamous cell carcinoma specimens (HLug-S030PG02) were purchased (OUTDO, Shanghai, China). Antibodies specific against E2A (sc-133074, Santa Cruz, Burlingame CA, USA) and p63 (CY5659, Abways, Shanghai, China), Ki67 (9449, CST, USA) and cleaved caspase-3 (CC3) (9661, CST, USA) were used for immunohistochemistry (IHC) staining. Immunohistochemical assay was performed as described²⁵ . Slides were scanned by NanoZoomer (Hamamatsu, Japan). E2A and p63 images were analyzed by calculating the integrated optical density (IOD) using Image-Pro Plus 6.0 (Media Cybernetics, MA, USA), and the average optical density (AOD) was calculated using the formula: AOD = IOD/Area as described⁵⁶ . Ki67 and CC3 images were analyzed by calculating the percentage of Ki67/CC3 positive tumor cell⁵⁷ .