Fig 1: High-throughput screening approach using the ß-TrCP reporter.a Schematic showing the ß-TrCP reporter fused to NanoLuc luciferase (NLuc). b Relative luciferase activity of MDA-MB-231 cells stably expressing ß-TrCP reporter treated with DMSO, MLN-4924, or cycloheximide (CHX) for 6 h. Graph is a box-and-whisker plot where the red line represents the median, the box represents the inter-quartile range, and the whiskers represent 5–95 percentiles. Dots represent individual data points beyond these percentiles. n = 32 replicates. c Workflow of the high-throughput screening (HTS) strategy. MDA-MB-231 cells stably expressing the ß-TrCP-NLuc reporter were seeded in 1536-well plates. The initial screen used the MIPE 5.0, and NPC 2.0 libraries comprising over 5000 approved and investigational drugs. After 6 h of treatment, luminescence was measured using a PHERAstar FSX microplate reader (BMG Labtech). d Scatter plot summarizing luminescence of MDA-MB-231 cells expressing ß-TrCP-NLuc reporter treated with compounds from MIPE 5.0 and NPC 2.0 library as described in (c) Each dot represents the percent activity of each small-molecule compound tested normalized to neutral and negative controls. Shaded regions highlight compounds demonstrating greater than 50% change relative to DMSO-treated controls. e STRING pathway analysis of proteins whose inhibitors were found to activate ß-TrCP activity in our screen. Nodes were colored green if they are a receptor-tyrosine kinase (RTK). f Relative reporter levels of MDA-MB-231 cells expressing either ß-TrCP-mVenus reporter, mutant ß-TrCP reporter, or Skp2-mCherry reporter treated with increasing doses of Sunitinib. Error bars represent SEM from n = 2 repeats. g Single-cell traces of ß-TrCP-mVenus reporter levels in MDA-MB-231 cells treated with Sunitinib at the indicated time. Data is median ± 95% confidence intervals. h Exponentially growing MDA-MB-231 cells were treated with Sunitinib (3 µM) for the indicated times. Whole-cell lysates were resolved in SDS-PAGE and immunoblotted for the indicated proteins. Representative blot of n = 3 independent experiments. i Immunoprecipitation of ß-TrCP with the SCF complex components Skp1 and Cullin1 in MDA-MB-231 cells treated with either DMSO or Sunitinib. Quantification of enrichment of bands compared to control is shown beneath the relevant bands. Representative blot of n = 3 independent experiments. j CCK-8 assay to determine percent growth inhibition of MDA-MB-231 cells treated with either control siRNA or ß-TrCP1,2 siRNA. Cells were treated with different doses of doxorubicin with or without 5 µM sunitinib. Error bars represent SEM from n = 4 experiments. k Model showing relative ß-TrCP activity across quiescence (G0) and different cycling phases. l Discovery of RTK signaling as a modulator of active SCF- ß-TrCP complex. Sunitinib malate promotes the association of ß-TrCP with core SCF complex components. Source data for all figure panels are provided as a Source Data file.