Figure 2.

High-Content Screening Reveals Significant and Dose-Dependent Reduction in MUC1 by the FDA-Approved SYK Inhibitor R406

(A) Screening pipeline.

(B) Primary screen revealed 4 major groups of compounds that affected MUC1 levels. MUC1 immunofluorescence (IF) signal intensity per cell (normalized to positive control JQ1 minus DMSO-treated controls) plotted versus DMSO-normalized cell number. Horizontal and vertical dashed lines delineate mean DMSO values ± 3∗SD for both MUC1 intensity and cell number. Lavender-shaded area demarcates candidate MUC1 suppressors.

(C) qPCR and cell viability profiling screens identified 4 compounds that reduced MUC1 protein abundance without changing MUC1 mRNA level, and in the absence of cytotoxicity for the previously identified active concentrations (the concentrations, which showed decrease in cell number in the initial screen, were removed from this analysis). Left: MUC1 signal intensity per cell plotted versus MUC1 mRNA level (qPCR assay). Both parameters are normalized to JQ1 minus DMSO-treated controls. Right: JQ1 minus DMSO-normalized MUC1 signal intensity per cell plotted versus DMSO-normalized cell viability (a number of viable cells after 6 days’ exposure to the test compounds). Horizontal dashed lines delineate SD for DMSO-treated control wells for both cell viability and MUC1 qPCR. Green-shaded areas demarcate candidate hits.

(D) R406 concentration response curves for MUC1 protein abundance (orange), MUC1 mRNA abundance (light gray), and cell viability (dark gray).

(E) MUC1 IF in kidney epithelial cells treated for 48 h in the absence (DMSO) and presence of R406 at the indicated concentrations.