ub cst 20326  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The protease cathepsin K can debulk the cancer glycocalyx

doi: 10.1016/j.jbc.2026.111206

Figure Lengend Snippet: Only cathepsin K degrades cell-surface mucins on K562s. A, schematic describing the flow cytometry assay used to evaluate the degradation of cell-surface mucins on K562 cells by cathepsins. B, cell viability of cells following enzymatic treatment. Normalized staining for ( C ) MUC1, ( D ) CD43, and ( E ) total mucins via StcE E447D following enzymatic treatments for 3 h at pH 6 in Hanks' balanced salt solution (HBSS). Staining was normalized such that PBS-treated and fluorescence minus-one controls are defined as 100% and 0% staining within each replicate ( n = 3–4 biologically independent replicates). Cathepsins have been labeled with their letter, for example, “A” refers to cathepsin A. CTSE was excluded because of the low pH of the CTSE activation buffer causing cellular toxicity . See for representative flow cytometry histograms, assays performed at pH 5 and 7, and bar graphs with raw median fluorescence intensity (MFI) values. Data are shown as mean ± SD from three to four biologically independent replicates, and each dot represents a single replicate. p Values determined via one-way ANOVA corrected via Dunnett’s multiple comparison test, with a single pooled variance. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. CTSE, cathepsin E; StcE E447D , inactive point mutant of StcE used as a pan-mucin probe.

Article Snippet: Mucin and MUC1 staining was performed with 15 μg/ml biotin-StcE E447D and 2 μg/ml anti-MUC1 antibody (Novus Bio, clone 967) in PBST for 1 h at room temperature, followed by 3 × 10 min wash with PBS-T. Then, the membrane was incubated with IRDye 800CW Streptavidin and 680RD Goat anti-Mouse IgG (LI-COR Biosciences) according to the manufacturer's recommendations, with 3 × 10 min wash with PBS-T before imaging.

Techniques: Flow Cytometry, Staining, Fluorescence, Labeling, Activation Assay, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The protease cathepsin K can debulk the cancer glycocalyx

doi: 10.1016/j.jbc.2026.111206

Figure Lengend Snippet: CTSK degrades cell-surface mucins across multiple cell lines. A, schematic describing the flow cytometry assay for detecting the degradation of cell-surface mucins on H82, OVCAR3, MCF10A, and MCF10 MUC1 cells. B, median fluorescence intensity (MFI) of MUC1 and total mucins via StcE E447D . C, normalized MFI of MUC1 staining for MCF10 ± MUC1 following enzymatic treatments for 1 h a 37 °C at pH 6.75 in Hanks' balanced salt solution (HBSS). D, normalized StcE E447D staining for cell lines following enzymatic treatment for 1 h a 37 °C at pH 6.75. See , A – D for representative histograms. Data are shown as mean ± SD from two to four biologically independent replicates, and each dot represents a single replicate. P Values determined via ( C ) two-way ANOVA or ( D ) one-way ANOVA, both corrected via Tukey’s multiple comparison test, with a single pooled variance for each cell line. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. CTSK, cathepsin K; MUC, mucin; StcE E447D , inactive point mutant of StcE used as a pan-mucin probe.

Article Snippet: Mucin and MUC1 staining was performed with 15 μg/ml biotin-StcE E447D and 2 μg/ml anti-MUC1 antibody (Novus Bio, clone 967) in PBST for 1 h at room temperature, followed by 3 × 10 min wash with PBS-T. Then, the membrane was incubated with IRDye 800CW Streptavidin and 680RD Goat anti-Mouse IgG (LI-COR Biosciences) according to the manufacturer's recommendations, with 3 × 10 min wash with PBS-T before imaging.

Techniques: Flow Cytometry, Fluorescence, Staining, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The protease cathepsin K can debulk the cancer glycocalyx

doi: 10.1016/j.jbc.2026.111206

Figure Lengend Snippet: Cathepsin K (CTSK) tolerates glycans near the cleavage site. A, cleavage motif of CTSK was generated from mass spectrometry analysis of ( left ) glycopeptides, ( center ) nonmodified peptides, and ( right ) modified and nonmodified peptides generated from CTSK digestion of purified and recombinant mucins and nonmucin glycoproteins, followed by trypsin digestion (see the section). The bar graphs on top of the glycopeptide cleavage motif indicate the frequency of O -glycosylation at each threonine and serine residue at that position. B, top, visualization of CTSK cleavage sites in recombinant MUC1 residues 24–47. The sialylated core-1 glycan at specific resides indicates that glycans were seen at those sites. This specific glycan was seen often in the dataset, but its depiction here is only intended to indicate glycosites, not to represent the diversity of all glycans detected at each glycosite in the dataset. Purple diamond , sialic acid; yellow circle , galactose; yellow square , N-acetylgalactosamine; and yellow circle , glycosylation site. Colored bars represent individual detected peptide sequences from CTSK cleavage only, with any shared peptides with chymotrypsin removed. Bottom, annotated spectrum for the indicated MUC1 O -glycopeptide. C, top, visualization of CTSK cleavage sites in recombinant P-selectin glycoprotein ligand-1 (PSGL-1) residues 148 to 197, represented as in ( B ), but this time from the tryptic + CTSK dataset, with all tryptic cleavage sites removed. Bottom, annotated spectrum for the indicated PSGL-1 O -glycopeptide. MUC, mucin.

Article Snippet: Mucin and MUC1 staining was performed with 15 μg/ml biotin-StcE E447D and 2 μg/ml anti-MUC1 antibody (Novus Bio, clone 967) in PBST for 1 h at room temperature, followed by 3 × 10 min wash with PBS-T. Then, the membrane was incubated with IRDye 800CW Streptavidin and 680RD Goat anti-Mouse IgG (LI-COR Biosciences) according to the manufacturer's recommendations, with 3 × 10 min wash with PBS-T before imaging.

Techniques: Generated, Mass Spectrometry, Modification, Purification, Recombinant, Glycoproteomics, Residue

Journal: The Journal of Biological Chemistry

Article Title: The protease cathepsin K can debulk the cancer glycocalyx

doi: 10.1016/j.jbc.2026.111206

Figure Lengend Snippet: Cathepsin K (CTSK) sheds bulky glycan polymers across multiple cell lines. A, schematic describing the flow cytometry assay for detecting shedding of glycan polymers from H82, OVCAR3, MCF10A, and MCF10 MUC1 cells following enzymatic treatments with CTSK, heat-inactivated CTSK (HI CTSK), StcE, heparinase, chondroitinase, and the polySia-specific endosialidase (EndoNA). Cells were stained for ( B ) heparan sulfate using fibroblast growth factor 2 (FGF2), which is a probe for heparan sulfate, ( C ) polysialic acid using anti-polySia antibody (clone 735), ( D ) chondroitin sulfate using anti–chondroitin sulfate antibody (clone CS-56), and ( E ) viability following enzymatic treatment of cells for 1 h at 37 °C at pH 6.75. Staining was ( B ) normalized median fluorescence intensity (MFI) to 100% buffer control and 0% secondary only or ( C and D ) quantified as percent positive staining because of the broad and non-normal distribution of the cell populations. See for normalized staining values and representative histograms. F, change in glycocalyx thickness of YSCCC, MCF10A, and MCF10 MUC1 cells following enzymatic treatment relative to buffer control was measured using scanning angle interference microscopy. Each data point is the average of 20 to 50 individual cell measurements performed on a single day and represents an independent biological replicate. See for individual cell measurements. Data are shown as mean ± SD from two to five biologically independent replicates, and each dot represents a single replicate. p Values determined via one-way or two-way ANOVA corrected via Tukey’s multiple comparison test, with a single pooled variance for each cell line. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Mucin and MUC1 staining was performed with 15 μg/ml biotin-StcE E447D and 2 μg/ml anti-MUC1 antibody (Novus Bio, clone 967) in PBST for 1 h at room temperature, followed by 3 × 10 min wash with PBS-T. Then, the membrane was incubated with IRDye 800CW Streptavidin and 680RD Goat anti-Mouse IgG (LI-COR Biosciences) according to the manufacturer's recommendations, with 3 × 10 min wash with PBS-T before imaging.

Techniques: Glycoproteomics, Flow Cytometry, Staining, Fluorescence, Control, Microscopy, Comparison