Ki 67 ma5 14520

A standard immunohistochemical analysis (IHC) protocol was performed on the tumor sections collected from the in vivo experiment. Briefly, tumor sections (5 µm thick) were dewaxed by xylene (5 min, 2x) and rehydrated with ethanol gradient (100%, 95%, and 70%, each for 5 min), and followed by blocking of endogenous peroxidase activity using 3% hydrogen peroxide. Antigen retrieval process was carried using a microwave (power set at high), while the slides were immersed in ethylenediaminetetraacetic acid (10 mM EDTA, pH 8.0) for 2 min and blocked with 10% normal goat serum. The sections were then incubated with primary antibodies SREBP1 (ab28481; 1:100 dilution), ZEB1 (ab228986; 1:100 dilution), vimentin (ab92547; 1:100 dilution) from Abcam (Abcam, MA, USA), E-cadherin (20874-1-AP; 1:100) from Proteintech Group (Proteintech, IL, USA), and Ki-67 (MA5-14520, 1:100 dilution) from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA) overnight in cold, followed by the incubation with secondary antibody of goat anti-mouse IgG HRP-conjugated (1:10,000) using a HRP Polymer Kit (#TP-015-HD; Lab Vision, Fremont, CA, USA). The slides were then stained with diaminobenzidine (DAB) and counterstained with Gill’s hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA).

Mouse (B16-F10) and human (A375 and A2058) melanoma cells were purchased from American Type Culture Collection (Manassas, VA, USA). These cells were cultured in DMEM (Invitrogen; Carlsbad, CA, USA) medium containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 mM glutamine, 100 mg/mL streptomycin (Invitrogen; Carlsbad, CA, USA) and 100 U/mL penicillin at 37 °C in 5% CO₂ atmosphere. The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): p-PTEN (sc-377573), PTEN (sc-7974), p-AKT (sc-293125), AKT (sc-5298), COX-2 (sc-70879), NFκB p65 (sc8008), MC1R (sc-28990). Antibody against β-actin (A5441) was purchased from Sigma–Aldrich (St. Louis, MO, USA). The antibody against Ki67 (MA5-14520) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The antibody against CD31 (NCL-L-CD31-607) was purchased from Leica Biosystems (Newcastle Upon Tyne, UK). The MTII peptide was purchased from Kelowna International Scientific Inc. (San Chung District, New Taipei City, Taiwan).