Extended Data Fig. 6: Calu-3 GOF cells express mucins that are sensitive to StcE digestion.
a. RT-qPCR analysis of target-gene expression for indicated GOF cell lines graphed as expression fold change over NTG for each gene. Statistical significance is measured by individual two-sided t-tests comparing GOF lines to NTG, n = 4 biological replicates. Dotted line indicates NTG expression level (normalized to 1). b. Western blot analysis of MUC1-, and MUC4-overexpressing Calu-3 cells. Bottom panels were imaged in a different channel for β-actin content as a loading control. One western blot was conducted in this experiment. c. Western blot analysis of CD44-overexpressing Calu-3 cells confirms protein upregulation. Treatment with StcE protease eliminates larger CD44 isoforms; two western blots were conducted with similar results. Bottom panels were imaged in a different channel for β-actin content as a loading control. One western blot was conducted in this experiment. d. Gating strategy for flow cytometry analysis, showing NTG Calu-3 cells treated with StcE protease. e. Flow cytometry histogram of CD44-GOF Calu-3 cells treated with StcE protease, stained with anti-CD44 antibody conjugated to PE. One experiment was conducted, CD44 guide 1 +StcE n = 12548, -StcE n = 21310, CD44 guide 2 +StcE n = 11296, -StcE n = 14280. f. Flow cytometry histogram of MUC1 GOF cells treated with StcE protease and stained with anti-MUC1 antibody demonstrates removal of MUC1 on the cell surface by StcE treatment. One experiment was conducted, MUC1 guide 2 isotype control n = 62687, and MUC1 guide 2 +StcE n = 33825, and -StcE n = 35500. For all panels: error bars denote mean ± s.e.m., significance is indicated as: n.s.=not significant, *= P < 0.05, **= P < 0.01, ***=P < 0.001, ****= P < 0.0001.
