Fig. 2: Linker-free Pro-BA outperforms the linker-containing degraders in vitro.

A H3122 cells were incubated with Pro-BA, Pro-PEG1-BA and Pro-PEG3-BA separately at 100 nM for 48 h. The expression levels of EML4-ALK and GAPDH were assessed by immunoblotting. B H3122 cell viability was measured via CCK-8 assays following a 48-hour exposure to Pro-BA, Pro-PEG1-BA, and Pro-PEG3-BA at various concentrations. Data is represented as mean ± SD of three independent experiments. C Summary of DC₅₀, D_max, IC₅₀ values, and molecular weight (MW) for the indicated compounds. D H3122 cells were exposed to Pro-BA, Pro-PEG1-BA, or Pro-PEG3-BA at 500 nM for 24 h, and cell cycle distribution was assessed using flow cytometry. E The bar graph illustrates the percentage of H3122 cells in the G1, S, and G2 phases as shown in (D). F H3122 cells were treated with Pro-BA, Pro-PEG1-BA or Pro-PEG3-BA at 500 nM for 24 hours, and apoptosis was assessed using flow cytometry. G The bar graph shows the percentages of early (left) and late (right) apoptotic cells, as indicated by the Q3 and Q2 quadrants in (F), respectively. H H3122 cells were treated with 5 μM Pro-BA or Pro-PEG3-BA for 5 h. UHPLC-MS/MS analysis of intracellular amounts of Pro-BA and Pro-PEG3-BA per 5 × 10⁶ cells. Data are presented as mean ± SD from three independent experiments, two-tailed Student’s t-test. For (E), (G), data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test (two-tailed). The gating strategies for flow cytometry analysis are shown in Supplementary Fig. 8. Source data are provided as a Source Data file.