Due to their high molecular weight, PROTACs can suffer from poor cell permeability and tools to precisely measure their cell membrane penetration are still being developed. Nonetheless, the catalytic mode of action of degraders may compensate for their lack of cell permeability since a low concentration of intracellular PROTAC may generate a substantial knockdown effect. To ensure that caged PROTAC 3 enters the cell before being uncaged, a washout experiment was conducted. After a 2 h-incubation with compound, HeLa cells were thoroughly washed to remove residual PROTAC then were irradiated at 365 nm for 60 seconds and further incubated for a few hours. Western blot analysis suggests that 3 effectively entered the cells within the 2 h-treatment period since degradation of BRD4 was still observed after irradiation (Figure ). Incomplete knockdown of BRD4 was noticed with PROTAC 4 after a 2-h incubation and correlated with the slower degradation rate seen in the time-course experiment (Figure ). BRD4 inhibition is reported to significantly impact cell proliferation, and we therefore examined the overall phenotypic effect of 3 in HeLa cells using live-cell imaging analysis before and after irradiation. First, PROTAC 3 was tested alongside pan-bromodomain inhibitor JQ1 without irradiation. Cell proliferation was checked over 24 h and compared to the cell count at the start of the experiment (Figure ). A 30% reduction in proliferation relative to vehicle (DMSO) was observed with 100 nM JQ1, while increasing concentrations (1 and 10 μM) resulted in minimal change. In the absence of irradiation, caged PROTAC 3 showed a dose-dependent effect on cell growth with 100 nM reducing cell proliferation by 20%, whereas 1 and 10 μM negatively affected cell count by 40% and 55%, respectively. This experiment strongly suggests that the caged compound 3 can effectively enter cells and engages a JQ1-dependent mechanism by inhibiting BRD4. PROTAC 2 and 4 also strongly impeded cell proliferation as compared with JQ1, emphasizing the difference between BRD4 inhibition and degradation (Supporting Information, Figure ).