galectin 3 gal 3  (R&D Systems)

Journal: Cancer Discovery

Article Title: Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer

doi: 10.1158/2159-8290.CD-22-0644

Figure Lengend Snippet: a2,6-SA and ST6GAL1 inhibit cluster formation and confer sensitivity to PAX. A, Left, flow profile of MDA-MB-231 cells with α2,6-SA–low and α2,6-SA–high populations. Right, representative images of sorted α-2,6-SA–low and α2,6-SA–high cells at 4 hours of clustering (top) and cluster formation curves (cluster size by area) of sorted cells (bottom). Scale bars = 60 μm. See Supplementary Videos S1 and S2 for cluster videos. B, Flow histograms of SNA-binding signals (left), representative cluster images (middle), and cluster formation curves (right) of MDA-MB-231 cells: ST6WT and ST6KO cells transfected with control vectors (-Con), and ST6KO cells transfected with ST6GAL1 overexpression vector (-OE). Scale bars = 100 μm. Cluster videos are available as Supplementary Videos S3–S5. C, Left: cell death (%) of CTC-092 PDX tumor cells ex vivo 24 hours after paclitaxel (PAX) treatments at 0, 25, 50, and 250 μg/mL, measured as DAPI positivity of live cells via flow cytometry (ns, not significant). Right, representative flow profile of DAPI and SNA signals in PAX-treated cells at the high dose of 250 μg/mL. D, Representative flow histograms (left) and SNA-high population (%) within alive CTC-092 cells (right) after 24 hours of PAX treatment at indicated doses (0–250 μg/mL). E, Representative images of CTC clusters formed at 24 hours (left) and the time-course cluster formation curves (right, cluster size) of CTC-092 PDX cells ex vivo upon treatment with PBS and PAX-NAB at 25 μg/mL. Scale bar = 50 μm. See Supplementary Videos S6 and S7 for cluster videos. F, Representative flow plots (left) and quantified viability (DAPI exclusion %; right graph) of flow sorting–enriched SNA-high and SNA-low MDA-MB-231 cells (as shown in A ) after overnight treatment with PAX at 0 and 25 μg/mL. G, Immunoblots of ST6GAL1, ST3GAL1, FUT3, CD44, and β-actin (loading control) of ST6WT and ST6KO cells at indicated time points (0, 24, and 48 hours) after PAX (25 μg/mL) treatment. Data are representative images of 2 biological replicates. H, Bright-field images of ST6WT and ST6KO cells (left) and their cell viability (%) after treatment with PAX at indicated doses (0–200 μg/mL; right). I, Cell viability of ST6WT and ST6KO cells after indicated time of paclitaxel (PAX) treatment at 25 μg/mL. P values were calculated with the Student t test in GraphPad unless otherwise indicated. Data, mean ± SD of 3–5 experimental replicates.

Article Snippet: Antibodies against ST6GAL1 (R&D Systems, AF5924), ST3GAL1 (R&D Systems, AF6905-SP), FUT3 (I; anti-CD174, BioLegend, 392602), FUT3 (II; Abcam, ab110082), CERCAM (Santa Cruz Biotechnology, D-4, sc-514083), Serpine-1 (Proteintech, 13801-1-AP), L1CAM (Thermo Fisher, 1.G11B1, MA526429), CD44 (Thermo Fisher, 8E2F3, MA515462), PODXL (Santa Cruz Biotechnology, 3D3, sc-23904), ICAM1 (Sigma, HPA004877), ECE1 (Santa Cruz Biotechnology, A-6, sc-376017), ALCAM1 (Proteintech, 21972-1-AP), CD97 (Santa Cruz Biotechnology, G-8, sc-166852), Na+/K+ ATPase aptha1 (Santa Cruz Biotechnology, C464.6), and β-actin (Sigma, A5441) were used as primary antibodies, and horseradish peroxidase (HRP)–conjugated secondary antibodies were from Promega (Rabbit W401B and Mouse W402B).

Techniques: Binding Assay, Transfection, Control, Over Expression, Plasmid Preparation, Ex Vivo, Flow Cytometry, Western Blot