Fig 2: Quantitative PCR and immunoblotting of five candidate gene products in DLBCL and LCL lines. (A) Reverse transcriptase and real-time PCR analysis of mRNAs for BMP8A, CCR6, HOXA9, NANOG and S100A8 in LCL and DLBCL (HBL-2, HT, SU-DHL-5, U2932 and U2940) cell lines. Data were normalized by the amount of GAPDH mRNA, expressed relative to the corresponding value for LCL, and are shown as means ± SD from triplicate data. (B) After quantification of Western blot intensity, the relative expression levels of each protein are shown with normalization of GAPDH. U2940 cells have the highest expression of the stem cell genes, HOXA9 and NANOG. Molecular weight listed in parenthesis. Error bars represent the standard error of the mean of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student t-test.

Fig 3: Spatial distribution of tumor marker candidate proteins in a HNSCC clinical specimen.(A) Pathological hematoxylin-eosin (H/E) staining of a fresh frozen tissue sample. Tumor region is marked. (B and C) MALDI MS image of S100A8 and S100A9 proteins in the healthy and tumor areas of the specimen. (D and E) MALDI MS image of unidentified protein examples of 4615 and 15126 Da.

Fig 4: S100A8/A9 purification of the cytoplasm of neutrophils. (A) Schematic overview of the purification process of S100A8/A9 from neutrophil cytosol. (B) The cytoplasmic fraction of neutrophils was isolated followed by 50% ammonium sulfate (AS) precipitation and anion-exchange chromatography (AEX). Indicated are the different peaks that were isolated (C2–C9). (C) Coomassie Brilliant Blue staining and (D) Western blot of isolated peak fractions, identifying S100A8 (red) and S100A9 (green) to be presented as mostly heterodimers and monomers. (E) Coomassie Brilliant Blue staining with a human serum albumin (HSA) calibration curve that was used to calculate the concentration of S100A8 and A9 heterodimers and monomers in the S100A8/A9 fraction used for functional experiments.

Fig 5: Protein expression and spatial distribution analysis of biopsy size regions of the tissue sample.(A) Healthy stroma and tumor region of interest are marked as ROI1 and ROI2 in the H/E stained section of the specimen, respectively. (B) Gel view format of image intensities of the detected proteins in the 4–17 kDa MW range. (C) Dual target intensities of pixels in the healthy (green) and tumor (red) ROIs are plotted for the 4615 Da protein and S100A8, (D) for the 4615 kDa protein and S100A9, (E) for the 15126 Da protein and S100A8, and (F) for the 15126 Da protein and S100A9.