Journal: British Journal of Cancer

Article Title: Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

doi: 10.1038/s41416-020-01093-z

Figure Lengend Snippet: a Organisation of ITIH5 domain structure labelled by amino acid (AA) position for ITI and Δs expression constructs expressed in the highly metastatic and low endogenous ITIH5-expressing S2-007 human PDAC cell line. Domain map adapted from Himmelfarb et al. Key features: cleavage site (CS); domains (grey): secretion signal peptide (SP), vault protein inter-α-trypsin domain (VIT), von Willebrand type A domain (vWA), multicopper oxidase domain (MCOD) and FLAG epitope (black). Labelled locations of ITIH5 and FLAG antibody binding. b Expression of full-length (ITI) and secretion-deficient ITIH5 (Δs) in S2-007 cells and immunoprecipitation (IP) from conditioned media to detect secreted ITIH5. ITIH5 antibody (binds on N-terminal side of cleavage site at AA578-606) and FLAG antibody (binds on the C-terminal side of CS after the end of ITIH5 open-reading frame). c Western blotting of extracellular vesicle (EV) preparations (particle-size range 20–300 nm, mean 155 nm measured using Nanosight TM particle analysis device) for ITIH5. Only minimal ITIH5 is present in EVs. d , e Western blotting of ITIH5 in cellular fractions. Two separately conducted cellular fractionations are shown per blot where either cytoplasmic fraction was separated from nuclear fraction or membrane/membrane-associated fraction was separated from cytoplasmic fraction. Antibodies used were the N-terminal-detecting ITIH5 (D) or C-terminal-detecting FLAG (E). Extracellularly, large and small fragments of ITIH5 are present. f ITIH5 is detected in the cytoplasm and nucleus using immunocytochemistry. Scale bar is 25 µm. g Locations of predicted ITIH5 nuclear localisation signals as predicted by sequence recognition algorithm. The algorithm is described in detail. , Na/K, sodium/potassium ATPase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PDAC cell lines (SUIT2 derivative S2-007, MiaPaCa-2 and PANC-1) were cultured in DMEM/F12 (Gibco, New York, USA, Cat #11330-032) with 5% foetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA, USA, Cat #S11195), 1% glutamine (Gibco, #25030-081), 0.25% non-essential amino acids (Gibco, #11140-050) and 1.5 and 1.0 μg/mL Puromycin (Gibco, #A11138-03) in a humidified incubator at 37 °C.

Techniques: Expressing, Construct, FLAG-tag, Binding Assay, Immunoprecipitation, Western Blot, Particle Size Analysis, Membrane, Immunocytochemistry, Sequencing

Journal: British Journal of Cancer

Article Title: Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

doi: 10.1038/s41416-020-01093-z

Figure Lengend Snippet: a Expression of full-length ITIH5 and secretion-deficient ITIH5Δs in metastatic human PDAC cell lines (S2-007 and MIAPaCa-2) correlates with rounded cell morphology and tight epithelial-like clustering. Expression of both ITIH5 and ITIH5Δs slowed cell motility in an in vitro wound healing/scratch assay S2-007, 8 h ( b ) and MIAPaCa-2, 72 h ( d ). c , e Quantification of the results from wound-healing assay measuring the percent of wound closure in S2-007 ( c ) and MIAPaCa-2 ( e ). * and ** S2-007 ITIH5 * P < 0.001 or ITIH5Δs ** P < 0.001 compared to control (Con) using Tukey’s test. Error bars show standard error. For MIAPaCa-2 cells, trends were similar although differences were not statistically significant.

Article Snippet: Human PDAC cell lines (SUIT2 derivative S2-007, MiaPaCa-2 and PANC-1) were cultured in DMEM/F12 (Gibco, New York, USA, Cat #11330-032) with 5% foetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA, USA, Cat #S11195), 1% glutamine (Gibco, #25030-081), 0.25% non-essential amino acids (Gibco, #11140-050) and 1.5 and 1.0 μg/mL Puromycin (Gibco, #A11138-03) in a humidified incubator at 37 °C.

Techniques: Expressing, In Vitro, Wound Healing Assay, Control

Journal: British Journal of Cancer

Article Title: Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

doi: 10.1038/s41416-020-01093-z

Figure Lengend Snippet: a Representative images of livers following intrasplenic injection of human PDAC cells (S2-007) transfected with control vector (Con), ITIH5 (ITI) or ITIH5Δs (Δs). White arrows highlight liver metastases. b Quantification of gross liver metastases from Con, ITI and Δs (median: 16, 10 and 6, respectively). c Quantification of metastasis size (mean length (cm) of the longest dimension of gross liver metastases) from Con, ITI and Δs (median: 2.65, 1.45 and 1.35, respectively) *** P < 0.001. d Quantification of liver metastasis (by liver weight) from Con, ITI and Δs (median: 1.66, 1.56 and 1.51, respectively). * P = 0.028. e Photomicrograph of representative H&E-stained sections of liver with metastases (dashed lines). f Photomicrograph of representative H&E-stained sections of lung from mice in ( a , e ). Black arrows highlight lung metastases. No lung metastases were identified in the ITIH5-injected mice. g Ratio of the area of liver metastases compared to the total histologic liver area. No significant differences. h Number of lung metastases in Con and Δs mice (ITI group not shown since there were no lung metastases). No significant differences. Error bars show standard error.

Article Snippet: Human PDAC cell lines (SUIT2 derivative S2-007, MiaPaCa-2 and PANC-1) were cultured in DMEM/F12 (Gibco, New York, USA, Cat #11330-032) with 5% foetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA, USA, Cat #S11195), 1% glutamine (Gibco, #25030-081), 0.25% non-essential amino acids (Gibco, #11140-050) and 1.5 and 1.0 μg/mL Puromycin (Gibco, #A11138-03) in a humidified incubator at 37 °C.

Techniques: Injection, Transfection, Control, Plasmid Preparation, Staining