Lamb3

Immunohistochemical (IHC) staining of the paraffin-embedded HNSCC tissue microarrays were conducted using primary antibodies against Slug (Cell Signaling Technology, 1:200), LAMB3 (Abcam, 1:250), and Podoplanin/gp36[EPR22182] (Abcam, 1:250), as previously described [22 (link)]. An isotype goat IgG antibody was used as a negative control. All slides were scanned with an Aperio ScanScope CS scanner (Vista, CA, USA). The histoscore quantification of each sample was analyzed by the Aperio Quantification software algorithms as previously described [23 (link)]. Histoscores of each slide, including ISH stain and IHC stain, were calculated according to the formula (3 + percent cells) × 3 + (2 + percent cells) × 2 + (1 + percent cells) × 1) total intensity/total cell number and were used to assess the histoscore of the pixel quantifications.

TMAs were created by Shanghai Outdo Biotechnology Co., Ltd. (Shanghai, China). IHC staining with antibodies against LAMB3, Ki-67, and PCNA (Abcam, MA, USA) was performed to detect protein expression levels following standard operating procedures. The positive staining scores were calculated by multiplying the percentage positive (0, < 5% of cells; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) by the staining intensity score (0, no coloration; 1, pale yellow; 2, yellow; and 3, dark brown) and were classified as follows: negative (0, −); weakly positive (1–3, + ); moderately positive (4–8, + + ); and strongly positive ( > 8, + + + ). We divided all patients into two groups (−/ + , low expression; and + + / + + + , high expression) and performed survival analyses.

Cells were lysed in lysis buffer containing 50 mM HEPES, pH 8.0, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl₂, 100 mM NaF, 10 mM Na₄P₂O₇·10H₂O, and a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Frozen tissue samples stored in liquid nitrogen were cut into pieces with scissors. Each sample was homogenized in lysis buffer at a ratio of 1:20. After centrifugation at 14,000 rpm for 20 min, the supernatant was collected. A BCA Protein Assay kit (Thermo Scientific, IL, USA) was used to measure total protein concentration. Aliquots (50 μg) of total cellular protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (6–15%) and electrotransferred onto Polyvinylidene Fluoride (PVDF) membranes, which were blocked with 5% skim milk at room temperature for 1 h. The membranes were then incubated with primary antibodies against the following proteins for western blot analysis: LAMB3, E-cadherin, and N-cadherin (1:1000; Abcam, MA, USA); phospho-Akt-S473, total Akt, vimentin, Slug, Snail, and β-actin (1:1,000; Cell Signaling Technology, MA, USA); and LAMA3 and LAMC2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following incubation with the corresponding secondary antibodies (1:5000; GE Healthcare, Buckinghamshire, UK), immune reactive bands were visualized by enhanced chemiluminescence (ECL) detection.