Epr5701

Western blots were performed as previously described [23 (link)] using specific antibodies against TP63 (EPR5701, Abcam), SOX2 (D6D9), SOX4 (Millpore), NOTCH1 (D1E11), NOTCH3 (D11B8), NANOG (D73G4), OCT4, KLF4 (D1F2), CD44 (EPR1013Y, Abcam), and ABCG2; GAPDH (1:2000, 14C10) and α-Tubulin (1:2000, 11H10) were used as internal references. All antibodies were purchased from Cell Signaling Technology, except where indicated, and used at 1:1000 dilutions. The full scans of the Western blots were quantified using the Alpha Innotech imaging software (San Leandro, CA, USA).

Collected fresh lung tissue resections were fixed in 4% paraformaldehyde, followed by dehydration and embedding in paraffin as routine protocol. Subsequently, the paraffin blocks were cut into 3-μm-thick sections and adhered to the slices for Multiplex fluorescent IHC staining. Briefly, the sections were firstly placed in a 65 °C oven for 1 h and deparaffinized in xylene, followed by rehydrated successively in 95%, 80%, and 70% alcohol. Then, antigen retrieval was performed by incubating in critic acid buffer at 95 °C for 20 min. Endogenous peroxidase was blocked by incubating in 3% H₂O₂ at room temperature (RT) for 10 min. Subsequently, 10% normal goat serum was added at RT for 1 h to block non-specific sites. Then, the sections were incubated with freshly diluted primary antibodies at RT for 1h. The primary antibodies that were used for cancer type validation included anti-p63 (Abcam, clone EPR5701, diluted at 1:12000), anti-TTF1 (Abcam, clone EP1584Y, diluted at 1:250), and anti-PGP9.5 (UCHL1) (Abcam, clone EPR4118, diluted at 1:250). Next, the second antibody was added to the sections and incubated at RT for another 1 h. Finally, the sections were incubated in freshly prepared fluorochrome at RT for 10 min. The antigen binding sites were visualized and analyzed with the PerkinElmer Vectra Automated Multispectral Imaging System.

Paraffin-embedded tumor tissues were cut into 4μm sections and processed for immunostaining. The sections were incubated overnight at 4°C with primary antibodies against TP63 (1:2000, EPR5701, Abcam), ΔNp63 (1:1000, N-16, Santa Cruz), Ki67 (1:1000, SP6, Abcam), SOX2 (1:200, D6D9, Cell Signaling Technology), CD44 (1:200, EPR1013Y, Abcam), KLF4 (1:200, D1F2, Cell Signaling Technology), and pan Cytokeratin (1:50, Novusbio), visualized using 3, 3-diaminobenzidine (DAB, Sigma-Aldrich) and counterstained with hematoxylin. Two senior pathologists who were blinded to the clinical data assessed and scored the IHC results. The fields were randomly selected for each sample under a light microscope with a 400× magnification. The staining index (SI) for TP63 (ΔNp63) was scored according to the staining intensity (0, no staining; 1, weak, light yellow; 2, moderate, yellow brown; 3, strong, brown) and the proportion of positive cells (0, 0%; 1, <10%; 2, <50%; 3, <75%; 4, ≥75%) per the following formula: SI= the proportion of positively stained cells×the staining intensity [57 (link)]. Cases with SI (TP63) >6 were classified into the high-expression group, and those with SI ≤6 were classified into the low-expression group. This scoring method was also used to evaluate the Ki67, SOX2, KLF4, and CD44 proteins expression in tumor xenografts.