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: Recombinant PSGL-1 (10 μg; Sino Biological; 13863-H08H), recombinant MUC1 (Sino Biological; 12123-H05H), recombinant CD43 (R&D Systems; 9680-CD-050), fetuin (Promega; V4961), and purified MUC16 (purified from OVCAR-3 as above) were mixed with CTSK or PBS control at a 1:50 CTSK:substrate molar ratio in 1:1 PBS:100 mM sodium acetate with a final pH of 5 (buffer pHed as above).

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