Hpa006881

Cells were seeded in 6-well plates containing coverslip in complete growth medium and, after treatment, fixed in 4% paraformaldehyde for 20 min at 20 °C, and permeabilized in 0.5% v/v Triton X-100 in PBS for 10 min, prior to be blocked with 5% v/v goat serum and 0.1% v/v Triton X-100 in PBS for 1 h. For ANXA2, permeabilization and blocking were performed with 0.2% w/v saponin in presence of 5% v/v goat serum and 0.2% w/v BSA in PBS, for 1 h. Immunofluorescence staining was obtained using anti-annexin A2 (1:125, ab41803, Abcam, Cambridge, UK), anti-NDRG1 (1:50, HPA006881, Sigma-Aldrich), anti-PARP1 (1:800, 46D11, Cell Signaling Technology, Danvers, MA), anti-STAT1 (1:100, HPA000931, Sigma-Aldrich) and anti-α-tubulin (1:200; clone DM1A, Merck Millipore, MA, USA), following overnight incubation at  4 °C. After washing, cells were incubated with secondary antibody anti-mouse Alexa Fluor-488 conjugate or anti-rabbit Alexa Fluor-488 and Alexa Fluor-567 conjugate (1:200) (Thermo Fisher Scientific), in the dark for 30 min at 20 °C. Coverslip was mounted onto slides using an antifade mounting reagent containing DAPI. Slides were observed under a fluorescence microscope (Leica Biosystems, Newcastle, UK) using a 100X oil immersion objective.

Slides of the OSCC cases (125 cases for the evaluation of neoplastic island proteins and 96 cases for tumor stroma) were incubated with anti-PGK1 (SAB1300102, Sigma) diluted 1:50, anti-NDRG1 (HPA006881, Sigma) diluted 1:100, anti-LTA4H (HPA008399, Sigma) diluted 1:100, anti-CSTB (HPA017380, Sigma) diluted 1:250, anti-COL6A1 (HPA019142, Sigma) diluted 1:1000, anti-ITGAV (HPA004856, Sigma) diluted 1:300 and anti-MB (HPA003123, Sigma) diluted 1:75 according to The Human Protein Atlas and the manufacturers’ instructions and were assessed using the Envision detection system (Dako). The control reactions were performed by exclusion of the primary antibodies. The specificities of the antibodies employed were shown by western blot with seven cell line extracts.