Figure 5. Development of a Catalytic Inhibitor of PRC1.
(A) Chemical structures of PRT4165 and GW-516.
(B) Acid-extracted histones from PC3 cells treated as indicated for 24 hours were subjected to immunoblotting followed by densitometry. H2AUb was normalized to control and the IC50 determined.
(C) PC3 cells were subjected to sphere assay in the presence of PRT4165 or GW-516. The inhibition of sphere formation was normalized to control and the IC50 determined.
(D) Histone H2A was incubated with RNF2 in the presence of E1 and E2. Reactions were carried out in the presence of PRT4165 or GW-516.
(E and F) Cells were treated with GW-516 or PTC209 at the indicated concentrations for 6 hours (E), or treated with GW-516 or PTC209 for the indicated times (F).
(G and H) Expression of RNF2 target genes upon RNF2 depletion or GW-516 treatment in PC3 (G) and RM1 cells (H). Bars, mean±SD.
(I and J) Representative images (I) and quantification of luciferase counts (J) of mice injected i.c. with PC3 cells and treated with vehicle (DMSO) or GW-516 as indicated. Mice were dosed twice per week. Bars, mean±SEM.
(K) Representative images (left, bar=50 μm) and percentages of staining intensity (right, Bars, mean±SD) of IHC staining for CCL2 or H2AUb of bone tissues from (I).
See also Figure S5.