Fl 1231

Standard immunofluorescence staining procedure was performed for STn antigen expression by anti‐STn mAb TKH2 and Vicia Villosa Agglutinin (VVA) in PDAC (T3M4 and Capan‐2) WT, SC and SC‐R cells. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.15% Triton X‐100 in 1× PBS containing 1% BSA and were incubated with mouse anti‐TKH2 (a kind gift from Dr Ulla Mandel, University of Copenhagen, Denmark) and fluorescein labelled VVA (FL‐1231, Vector Laboratories) for 2 hours at room temperature. Cells incubated with mAb TKH2 were washed and stained with Alexa Fluor 488‐conjugated goat antimouse IgG (Jackson ImmunoResearch). Finally, cells were washed and mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) and images were captured using Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss, Inc).

These experiments were performed as we previously described^{21 (link)–23 (link)}. For Western blot, antibodies for GAPDH (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cosmc (sc-67480; Santa Cruz Biotechnology), CEL (ab79131; Abcam, Cambridge, UK; sc-34883; Santa Cruz Biotechnology), DMBT1 (MAB59151; R&D Systems, Minneapolis, MN, USA), elastase (ab21593; Abcam) and trypsin (ab166898; Abcam) were used. Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. For immunohistochemistry, antibodies for Tn Antigen (MA180055; Thermo Fisher Scientific, Grand Island, NY, USA), STn Antigen (ab115957; Abcam), insulin (8138; CST, Beverly, MA, USA) and VVA-fluorescein (FL-1231; Vector Laboratories) were used at a dilution of 1:100^{23 (link)}. Ten micrograms per milliliter of Sambucus nigra lectin (SNA) (B- 1305–2; Vector Laboratories, Burlingame, CA, USA) was complexed with 1 µg streptavidin-HRP. For real-time PCR, RNA was extracted with the RNeasy Plus Tissue Mini Kit (Qiagen).