Cotl1

Western blots were generated according to a previously described protocol [28 (link)]. Briefly, 5 µL protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. Rose-Anne Romano) [29 (link)], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Tris-buffered saline with 0.05% Tween-20. Protein expression was detected with the LumiGLO peroxidase chemiluminescent substrate kit (SeraCare, Milford, MA, USA), and membranes were imaged using a Bio-Rad ChemiDoc imaging system. Uncropped Western blot images can be found in Figures S7–S9.

Tissue and cells were collected after washing with cold PBS and then lysed in RIPA lysis buffer (Solarbio) with protease inhibitor cocktail (Roche). Protein concentration was determined with a Lowry protein concentration detection kit (Solarbio). An equal amount of proteins for each sample was subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. Antibodies were GATA3 (1:500, Proteintech), COTL1 (1: 200, Proteintech), N-Ras (1:500, Proteintech) and GAPDH (1:1000, Proteintech).

De-identified SCC patient samples were obtained from the archives of the Department of Oral and Maxillofacial Pathology, the University of New York at the University at Buffalo. An oral cavity tumor with a normal tissue microarray (US Biomax, Inc.—Tissue Array, T271b, Derwood, MD, USA) and SCC tissue slides were deparaffinized and sequentially rehydrated in decreasing concentrations of ethanol in water. After heat-induced antigen retrieval in sodium citrate, tissues were blocked and then incubated overnight with primary antibodies specific to p63 (4A4, 1:300), COTL1 (Proteintech, 1:200), or K14 (1:300), according to standard protocols. For immunofluorescence, the slides were incubated with appropriate fluorescent secondary antibodies, and coverslips were mounted with a mounting medium containing DAPI (Vector Laboratories, Newark, CA, USA) to stain nuclei, and then they were sealed for imaging. For immunohistochemistry, antibody labeling was visualized with an Impact DAB substrate kit (Vector Laboratories). Counterstaining was conducted using hematoxylin (Vector labs) before the slides were rinsed in tap water, air dried, and coverslipped with Permount mounting medium (Thermo Fisher Scientific, Waltham, MA, USA).