Supplementary Figures 1 - 9 from Quantitative Immunofluorescence Reveals the Signature of Active B-cell Receptor Signaling in Diffuse Large B-cell Lymphoma

PDF file, 871K, S1 Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S2 Schematic illustration of Tissuequest analysis workflow. Individual cells are first identified by finding the nuclei (DAPI); S3 Qualitative Western blot of whole cell lysates for untreated (BCR-) and crosslinked (BCR+) cell lines; S4 S4 Chromogenic staining (DAB, brown) of two paraffin-embedded patient DLBCL specimens with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S5 (A) Schematic illustration of Tissuequest workflow extended to subcellular localization of FOXO1. In this example there are 83% of all cells that are positive for FOXO1 and 67% of the cells show cytoplasmic FOXO1 staining (67%). The ratio of these percentages is used to calculate the Fcyt score; S6 (A) Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti- FOXO1; S7 (A) IHC staining of paraffin-embedded DLBCL cell lines U2932, DHL6 and Toledo, untreated and crosslinked, with anti-pAKTsub (red) and DAPI (blue); S8 (A) Comparison of immunofluorescent images evaluated by an expert using a 3-tier system (0 - negative, 1 - weak positive, 2 - strong positive); S9 Comparison of FOXO1 cytoplasmic localization and BCR signaling in primary DLBCL tumors.