(A) Structure of 1a-CA-biotin; the chlorambucil moiety (CA) forms a covalent bond with the cellular target once the small molecule is bound. Biotin is used to isolate biomolecule-small molecule adducts from cells.

(B) Normalized enrichment of r(GGGGCC) 66 from biomolecules isolated with streptavidin beads as compared to 18S rRNA determined by qRT-PCR. Cells were treated with 1a-CA-biotin alone or with 1a, 2, or 3 (competitive profiling). In competitive profiling experiments, the unreactive compound inhibits reaction of the target with 1a-CA-biotin and thus the target is depleted in pull-down fractions. Data presented as mean ± SD (n = 2).

(C) Cells overexpressing 66 (GGGGCC) repeats (C9-66R), but not 2 (C9-2R) or 20 (C9-20R), express poly(GP) and poly(GA) proteins. Asterisks demark nonspecific bands.

(D) Western blot and densitometry of poly(GP) and poly(GA) proteins in (GGGGCC) 66 -expressing cells treated with DMSO or compounds 1a, 2, or 3 (100 μM, 24 hr). Data represent mean + SEM (n = 3).

(E) RNA foci (red) are detected in the nucleus (Hoechst; blue) of C9-66R cells.

(F) Evaluation of the percentage of foci-positive cells by RNA FISH posttreatment. Data represent mean + SEM in ten fields.

(G and H) Compound 1a decreases RAN translation of poly(GP) proteins from sense (GGGGCC) 66 repeats in C9-66R cells (G) but does not influence levels of poly(GP) or poly(PR) proteins RAN translated from antisense (CCCCGG) 66 repeats in C9-AS-66R cells (H), as assessed by GP and PR immunoassays. Data represent mean + SEM (n = 3 or 4).

(I) The percentage of r(CCCCGG)-containing foci in C9-AS-66R cells is not affected by 1a.

(J) 1a decreases RAN translation of poly(G) in cells expressing (CGG) 88 upstream of GFP. Data presented as mean + SEM (n = 3).