Supplementary Figure S3 from Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6

Fig. S3. MUC16 expression in PDAC cells and PDAC patients’ samples using huAR9.6 mAb. (A) Western blotting for MUC16 (with huAR9.6) in normal HPDE cells and AsPC-1, T3M4, Capan-2, Panc-1, MiaPaCa-2, Capan-1, BxPC-3, S2-013, FG and SU86.86 PDAC cells. Loading control: α-tubulin. (B) Immunofluorescent staining for MUC16 (AF647-huAR9.6, red) and α-tubulin (AF488, green) in T3M4-WT and T3M4-MUC16KO cells. (C) Representative image of immunofluorescent staining for MUC16 (AF647-huAR9.6, red) in normal pancreas (n=7) and PDAC tissue (n=32) obtained from the Rapid Autopsy Program at UNMC. (D) Representative images of immunohistochemistry for MUC16 on a patient-derived xenograft (PDX) microarray using huAR9.6; (E) Graphical representation of varying histoscores; (F) Graphical representation of consistency between histoscore (huAR9.6-based MUC16 detection) and RNA sequencing of MUC16 gene expression in PDX microarray samples (n = 31). (G) Western blotting for MUC16 (with huAR9.6) in ascites fluids (n = 34) obtained from PDAC patients via the Rapid Autopsy Program at UNMC. Western blot of N-glycanase, O-glycanase and sialidase treated (H) T3M4 cell lysates and (I) MUC16 TR1.2 glycopeptides probed with huAR9.6 mAb. Detection of α-tubulin served as the loading control.