Aldh1

The methods and processes of immunohistochemical staining were performed as previously described 20 (link). Briefly, all OSCC tissue microarrays were cut into 4-μm sections. Slides were deparaffinized by dimethylbenzene and hydrated by ethyl alcohol sequentially. Antigen retrieval was conducted using sodium citrate (pH= 6.0) under high pressure. Then, the sections were incubated to block endogenous peroxidase activity using hydrogen superoxide. After blocking with goat serum at 37°C for 20 min, the slides were incubated with the following antibodies: ME2 (Cell Signaling Technology, USA), Slug (Cell Signaling Technology, USA), SOX2 (Cell Signaling Technology, USA), and ALDH1(Cell Signaling Technology, USA) at 4°C overnight. After washing the slides with PBS, an appropriate secondary biotinylated immunoglobulin G antibody solution and an avidin/biotin/peroxidase reagent were added to the slides. Immunohistochemical staining was accomplished with diaminobenzidine and then gently counterstained with hematoxylin (Invitrogen, Waltham, MA, USA).

Cells were lysed in Radioimmunoprecipitation assay buffer (Solarbio) containing phenylmethylsulphonyl fluoride protease inhibitor. Protein samples were subjected to 10% SDS polyacrylamide gel electrophoresis. The separated proteins were subsequently transferred to a polyvinylidene fluoride membrane. The membrane was blocked using 10% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. Primary antibodies recognizing the following proteins were used: CD24 (1:1000, Proteintech, Wuhan, China, Cat#18330-1-AP), SOX2 (1:1000, Cell Signaling Technology, Cat#14962), ALDH1 (1:1000, Cell Signaling Technology, Cat#54135), OCT4 (1:1000, Wanlei, Shenyang, China, WL02020), BMI1 (1:1000, Cell Signaling Technology, Cat#6964), YAP1 (1:1000, Cell Signaling Technology, Cat#14074), GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) (1:20000, Proteintech, Cat#60004-1). anti-phospho Histone H2A.X (Ser139) (1:1000, Cell Signaling Technology, Cat#9718). Thereafter, the membranes were incubated with secondary antibodies corresponding to the primary antibodies for 1 h at room temperature. Finally, the immunoreactive protein bands were visualized using ECL (NCM Biotech, Suzhou, China) Western blot detection reagent.

The 5 μm thick paraffin sections were deparaffinized in xylene and rehydrated in ethanol at different gradients (100%, 100%, 95%, 70% in sequence). Tissue slices were incubated in 3% H₂O₂ for 20 min to inactivate endogenous peroxidase. After being heated in 10 mM citrate buffer for 15 min, tissue sections were incubated with primary antibodies ER (21244-1-AP, 1:200; Proteintech, Rosemont, IL, USA), HER2 (60311-1-Ig, 1:200; Proteintech), ALDH1 (#54135, 1:200; Cell Signaling Technology, St Louis, MO, USA) and E-Cadherin (#14472, 1:200; Cell Signaling Technology) overnight at 4°C. Corresponding secondary antibody (#8114; Cell Signaling Technology) was added and incubated for 1 hour at the room temperature. Images were observed with Pannoramic MIDI (3DHISTECH Ltd, Budapest, Hungary).

Western blotting was performed according to a standard protocol, as described previously [16 (link)]. The total proteins were collected using SDS lysis buffer (Beyotime, Shanghai, China, P0013G), and protein concentrations were determined by Bicinchoninic Acid (KeyGen, Nanjing, China, KGP902). Nuclear extracts were obtained using the NE‐PER Nuclear and cytoplasmic extraction reagents (Thermo Scientific, Waltham, MA, USA, 78833) according to the manufacturer’s instructions. The following primary antibodies were used: FUBP1(ABE1330) from Merck Millipore (Boston, MA, USA); CD133 (#86781), ALDH1 (#54135), CD44 (#37259), p‐GSK3β (Ser9) (#9323), GSK3β (#9832), n‐p‐β‐catenin (Ser33/37/Thr41)(#8814), β‐catenin (#9582), Histone (4499), and c‐Myc (#13987) from Cell Signaling Technology (Boston, MA, USA); LGR5 (ab75732) and DVL1 (ab233003) from Abcam (Cambridge, UK); and β‐actin (A5441) from Sigma‐Aldrich (St. Louis, USA). HRP‐conjugated anti‐rabbit IgG (Cell Signaling Tech, #7074) and anti‐mouse IgG (Sigma‐Aldrich, AP308P) were used as secondary antibodies. Proteins were determined using ECL Plus Reagent (Millipore, WBKLS0100).

Monoclonal antibodies against HSD17B2 (#TA504616), HSD17B3 (#CF811500), SHBG (#TA507187), and SRD5A1 (#PA5-75675) were obtained from Thermo Fisher Scientific Inc. (Bartlesville, OK, USA), while KLF4 (#12173), OCT4A (#2840), MRP1/ABCC1 (#14685), MDR1/ABCB1 (#13342), and ALDH1 (#36671) were all purchased from Cell Signaling Technology (CST, Beverly, MA, USA), and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dutasteride (#SML1221, ≥98% (HPLC)) was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).