Fig. 2.
Complete Freund’s adjuvant (CFA) enhances bromodomain-containing protein 4 (Brd4) coupling with acetylated voltage-gated sodium channel 1.7 (Nav1.7) promoters to enhanced Nav1.7 expression in dorsal root ganglia (DRG) neurons. (A) Chromatin immunoprecipitation–quantitative polymerase chain reaction (PCR; ChIP)–quantitative (q) PCR assay demonstrating CFA increased the abundance of Nav1.7 promoter fragments immunoprecipitated by the H3-specific antibody in DRG at 1 day after injection. **P < 0.01 versus saline 1d; n = 6. (B) and (C) Intrathecal Brd4-specific small interfering (si) RNA (Brd4 RNAi + CFA 1d, 10 μl, 5 μg) and JQ1 (CFA 1d + JQ1, 10 μl, 100 μM) injections both inhibited CFA-enhanced expression of Nav1.7 mRNA and protein in DRG. IB = immunoblotting; MS RNAi = missense siRNA; Veh = vehicle. JQ1 is an inhibitor of bromodomain and extraterminal (BET) binding to acetylated histones. **P < 0.01 versus saline 1d; #P < 0.05, ##P < 0.01 versus CFA 1d; n = 6. (D) Intrathecal Brd4-specific siRNA (Brd4 RNAi + CFA 1d, 10 μl, 5 μg) and JQ1 (CFA 1d + JQ1, 10 μl, 100 μM) injection both inhibited CFA-increased Nav1.7 promoter fragments immunoprecipitated by the Brd4-specific antibody. **P < 0.01 versus saline 1d; ##P < 0.01 versus CFA 1d; n = 6. (E) Representative images and statistical analysis demonstrating CFA (II) markedly increased Brd4-positive (red), Nav1.7-positive (green), and Brd4–Nav1.7 double-labeled (yellow) immunofluorescence in DRG neurons that were all inhibited by Brd4-specific siRNA (Brd4 RNAi + CFA 1d, 10 μl, 5 μg, III). JQ1 injection (CFA 1d + JQ1, 10 μl, 100 μM, IV) inhibited CFA-increased Nav1.7-positive (green) and Brd4–Nav1.7 double-labeled (yellow) but not Brd4-positive immunofluorescence in DRG neurons. **P < 0.01 versus saline 1d; ##P < 0.01 versus CFA 1d; n = 7. Scale bar = 50 μm. Thickness = 16 μm.