Figure 4: Cellular Activity of CBX2 ChD Ligand.

A) Relative cytosolic access of SW2_152F conjugated to a chloroalkane (SW2_152F-CA) was evaluated using chloroalkane penetration assay (CAPA). The half maximal cell penetration value (CP₅₀) of SW2_152F-CA was determined for SW2_152F-CA and CBX8 inhibitor SW2_110A-CA. B) Chemoprecipitations from HEK293T nuclear lysates using biotinylated SW2_152F (SW2_152F-B) were analyzed using immunoblot analysis. C) Analysis and quantification of SW2_152F in abrogating bulk binding of endogenous CBX proteins to chromatin in HEK293T cells by Sequential Salt Extraction. D). Chromatin immunoprecipitation (ChIP) from K562 cells using antibodies against IgG, H3K27me3, CBX2 and CBX8 was followed by quantitative PCR of genomic regions near Tm9SF4 (negative locus), TCF21, Fyn-2, and PAX7. Cells were treated with 10 μM SW2_152F for 4h prior to harvest. E) Chromatin immunoprecipitation (ChIP) in Hs68 fibroblast cells using antibodies against IgG, CBX7, and CBX8 followed by quantitative PCR of genomic regions at LMNB2 (negative locus), CCND2, and RUNX3. Cells were treated with 100 μM SW2_152F for 4h prior to harvest. For all qPCR, error bars represent SEM n=3 biological replicates, p-values were calculated using two-tailed Student’s t-test, * = p < 0.05, ** = p <0.01, *** = p < 0.001, **** = p <0.0001.