Secondary screen (Hit validation)

All successful hits from the primary screen in either cell line (104 proteins, 83 from hTERT-HMEC, 21 from MCF7, Supplementary Table 2) were re-screened using an almost identical methodology and controls, though some modifications were made. Of note, while secondary screens were performed in the same cell stocks as the primary screen, secondary screens were performed in the labs at the MRC LMS which may account for differences in the fraction of EdU negative cells identified compared to the primary screen (Supplementary Table 1 versus Supplementary Tables 3, 4). However, despite these differences, each screen was compared to controls done on the same day to control for any differences in population growth. Due to limited protein availability, the 21 MCF7 hits were re-screened across 5 doses in both cell lines (Supplementary Table 3), whereas the 83 hits from hTERT-HMEC were only re-screened in hTERT-HMEC cells (Supplementary Table 4). In the secondary screen, on day 1, each protein was diluted in PBS from their stock concentrations (1:20, 1:40, 1:80, 1:160 and 1:320) using the automated liquid handler Opentrons OT to form a dose curve which was dispensed onto cells manually. The Palbociclib control was also titrated (1, 0.5, 0.25, 0.125, 0.0625 μM) while the PBS amount added remained constant. The remaining workflow remained unchanged, however, washing was performed with a Biotek 50TS microplate washer, widefield imaging was performed using an Operetta CLS (PerkinElmer) with a 20X objective NA 0.8, and an identical image analysis method was conducted using Harmony software (PerkinElmer). Hits were determined using the same parameters as our primary screen and are listed in (Supplementary Table 5).

Purified ICAM3 and ICAM5 were purchased from Abcam (ab276251 and ab276709, respectively) and were diluted in PBS. Cell treatment was as in the secondary screen.