Journal: Nature biotechnology
Article Title: Design of a mucin-selective protease for targeted degradation of cancer-associated mucins
doi: 10.1038/s41587-023-01840-6
Figure Lengend Snippet: a-b, 4T07MUC1 cells (a) and OVCAR-3 cells (b) were treated with 50 nM StcE for 2 hours, washed 1x with 2 mM EDTA followed by 5x with DPBS, then cultured for the indicated times. Cells were then lysed and subjected to Western blotting for MUC1 (a) and MUC16 (b). Mucin bands are denoted by black arrows. c, Bioluminescent imaging of animals described in Fig. 4e. d, Total flux measurements quantified from (c). e, Plot depicting mouse masses of animals described in Fig. 4e. f-g, H&E staining of lungs from PBS-treated (f) or αHER2-eStcE treated (g) animals (n = 7 animals per group, 2 slides per animal). Percent area of lung metastases is quantified in Fig. 4g. h, Quantification of images from Supplementary Fig. 6 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the cytosol. i, Quantification of images from Supplementary Fig. 7 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the cytosol. j, Quantification of images from Supplementary Fig. 8 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the nucleus. k, Treatment regimen for BALB/c mice injected intravenously (I.V.) via tail vein with 4T07MUC1, HER2 cells. Doxycycline was included in the chow for the duration of the experiment to maintain MUC1 ectodomain expression. αHER2-eStcE at 10 mg/kg or an equimolar quantity of αHER2-eStcEE447D or αGFP-eStcE were injected I.V. every other day starting on day 0 (n = 9 animals per group). l, Total flux of the indicated days normalized to the total flux on day 0 for each mouse quantified from Supplementary Fig. 10. Data are mean ± s.e.m. P-values were determined using two-tailed Mann-Whitney test (d-e), two-tailed unpaired t-test (h-j), or Tukey-corrected one-way ANOVA (l). *p < 0.05, **p < 0.005, ***p < 0.0005.
Article Snippet: For recombinant human MUC16 digestion experiments, recombinant MUC16 (R&D Systems) was left unlabeled but was otherwise treated as described above.
Techniques: Cell Culture, Western Blot, Imaging, Staining, Injection, Expressing, Two Tailed Test, MANN-WHITNEY