Ab16700

Primary endometrial stromal cells from women with adenomyosis were isolated according to a protocol with minor modifications (25 (link)). Briefly, endometrial tissues were homogenized and treated in sequential steps of pancreatin-0.05% trypsin enzymatic solution, collagenase 4 (0.1 U/ml), and DNase I (16 μg/ml) solution in Ca2+/Mg2+-free PBS (Gibco^® Thermo Fisher Scientific, Sweden) and incubated for 30 minutes at each step. Enzymatically digested cell suspensions were filtered through a 100 µM cell strainer to remove larger debris while collecting the flowthrough containing both the epithelial and stromal fractions. Later, these cell suspensions were adherent and expanded in vitro for two-three generations, frozen, and stored in liquid nitrogen for the following experiments. The isolated ESC was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin solutions (Gibco, USA). When the cells reached 80-90% confluency, the cells were passaged, and the medium was changed every 2-3 days. Cyto-immunofluorescent staining for vimentin (Abcam, ab16700) and pan-cytokeratin (Abcam, ab86734) was applied for confirmation of stromal cell phenotype.

Proteins were extracted in RIPA buffer supplemented with complete, EDTA-free protease inhibitor cocktail (Roche Diagnostics) and phosphatase inhibitor cocktail 3 (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The following antibodies were used: β-actin (dil. 1/5000, A5441, Sigma), phospho-Smad2 (ser465/467) (dil. 1/1000, #3108, Cell Signalling), Smad2 (dil. 1/1000, #3102, Cell Signalling), E-cadherin (dil. 1/10000, BD610181, BD Pharmingen), α-smooth muscle actin (dil. 1/1000, A5228, Sigma) and vimentin (dil. 1/1000, Ab16700, Abcam). Uncropped blots are presented in the Source data file.

When cells reached about 80% confluence, they were treated with trypsin and removed from the third row of wells to a new 6-well plate. After repeated adherence and culture, the relatively purified OSCC cells were obtained.
The identification of OSCC cells was performed. In short, immunohistochemistry was applied to stain keratin, Vimentin, CSC-related identification, and isolation markers BMI1, ALDH1, and CD44. Slides containing OSCC cells were prepared on the strictly sterilized coverslips. The staining was performed following the manufacturer’s instructions of the streptavidin peroxidase (SP) immunohistochemistry kit, with polyclonal antibodies purchased from Abcam (Cambridge, UK) used as the primary antibodies: keratin (1:1000, ab155478), Vimentin (1:1000, ab16700), BMI1 (1:1000, ab155478), ALDH1 (1:1000, ab155478), and CD44 (1:1000, ab155478). Positive and negative controls were prepared during staining. Known positive cell slides were used as the positive control, while PBS was used to replace the primary antibody as the negative control (NC). The cytoplasm and/or cell membrane showing tan-stained particles was considered positive.