Fig 2: Development of electrophilic kinase degraders dependent on the SNAr covalent warhead.A. Structures of electrophilic promiscuous kinase degraders ZZ7-18-043, ZZ7-18-045, and a negative control of ZZ7-18-045 with slight chemical modification that negate SNAr reactivity. B. Western blots of kinases degradations in MOLT-4 cells treated for 3 hours with 1 μM indicated compounds. C. Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 hours treatment with 1 μM ZZ7-18-045. D. Western blots of LIMK2, Aurora A, and Aurora B degradations in Jurkat cells treated with the indicated inhibitors for 1 hour and then ZZ7-18-045 for 3 hours. E. Structures of electrophilic CDK4/6 degrader ZZ7-18-084 and negative controls with slight chemical modifications that negate SNAr reactivity. F. Western blots of CDK4 and CDK6 degradations in Jurkat cells treated with 1 μM indicated compounds for 6 hours. G. Quantitative proteome-wide mass spectrometry in MOLT-4 after 3 hours treatment with 1 μM ZZ7-18-084. H. Western blots of CDK4 and CDK6 degradations in Jurkat cells treated with the indicated inhibitors for 1 hour and then ZZ7-18-084 for 6 hours.

Fig 3: Sensitivity to CDK2 inhibitor tagtociclib at varying doses of abemaciclib.Lipo246 cells were cotreated with the CDK4/6 inhibitor abemaciclib (control, 10 nM, and 50 nM) and a range of CDK2 inhibitor concentrations (0, 0.25, 0.5, 1, 2.5, 5, 10 μM tagtociclib). (A) Proportion of pRB/RB positive cells following treatment with CDK2 inhibitor, tagtociclib. (B) Fold changes in the proportion of pRB/RB positive cells under treatment with tagtociclib. For each abemaciclib condition, fold changes were computed by normalizing the proportion of pRB/RB positive cells to the average proportion of the first three doses of tagtociclib (0, 0.25, and 0.5 μM). Error bars represent the SD across three technical replicates. Dashed lines indicate (A) the proportion or (B) fold change of pRB/RB positive cells without treatment with the CDK2 inhibitor.

Fig 4: Operating room-to-lab single-cell proteomic workflow using iterative indirect immunofluorescence (4i)(A) Experimental workflow of the operating room-to-lab pipeline used for single-cell, multiplexed immunofluorescence profiling. Lipo246 cells were cultured and dedifferentiated primary tumor cells were resected and dissociated to be treated with increasing concentrations of abemaciclib for 24 h. Cells were fixed and subjected to iterative indirect immunofluorescence imaging (4i) consisting of iteration of antibody labeling (block → 1°/2° Ab → elution) and imaging. Images from all 4i rounds are processed through a computational pipeline to perform single-cell segmentation, signal quantification, and feature extraction. (B) Lipo246 and primary tumor cells were stained for 17 cell cycle regulators pRB, RB, Ki-67, CDK2, CDK4, cyclin D1 (cycD1), cyclin E1 (cycE1), Cdt1, E2F1, cyclin A2 (cycA2), cyclin B1 (cycB1), p21, Mdm2, pS6, S6, p53, p16, and integrated DNA. Representative immunofluorescence images from sequential rounds of 4i demonstrate a subset of these regulators for repeated labeling of distinct protein targets within the same cells. Nuclear DNA staining is used for segmentation, image registration, and integration across imaging rounds. (C) Single-cell matrix of 4i-measured cell cycle features. Heatmap of normalized protein expression across all quantified regulators, with individual cells ordered by increasing ratio of phosphorylated to total RB (pRB/RB), computed from 4i measurements. The corresponding single-cell pRB/RB distribution is shown at right and exhibits a bimodal structure used to define low pRB/RB (blue; nonproliferating) and high pRB/RB (pink; proliferating) cell populations. This organization reveals coordinated variation in cell cycle regulators across the pRB/RB-defined axis of cell cycle state.