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. 2018 Mar 15;103(7):1124–1135. doi: 10.3324/haematol.2017.187013

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Figure 8. — ET-1 induced activation of ET_A receptors leads to the upregulation of Nav1.8 and activation of NF-κB in HbSS DRG neurons. (A) Cultured HbAA DRG neurons exposed to 400 nmol of ET-1 peptide for 24 hr displayed an increase in Nav1.8 protein. Co-administration of ABT-627 (1 μmol) prevented this increase. Left: Representative western blots. Right: Summary of densitometric analysis. n=3 biological repeats. **P<0.01 vs. the vehicle (Veh) group. (B) Cultured HbAA DRG neurons exposed to 400 nmol of ET-1 peptide for 90 minutes displayed an increase in phosphorylated p65 (p-p65). Co-administration of ABT-627 (1 μmol) 10 minutes prior to ET-1 stimulation prevented the increase in phosphorylated p65 protein. The amount of total p65 protein did not change among the treated groups. Left: Representative western blots. Right: Summary of densitometric analysis n=3 biological repeats. **P<0.01 vs. the vehicle (Veh) group. (C) The levels of phosphorylated p65 (p-p65), increase in the L3-L4 DRG of the vehicle-treated HbSS mice. This increase is not seen in the ABT-627-treated HbSS mice. Left: representative Western blots. Right: a summary of densitometric analysis. n=3 mice/group. *P<0.01 vs. the vehicle-treated HbAA group, ##P<0.01 vs. the vehicle-treated HbSS group. (D) 45 mins after intraperitoneal injection of the NF-κB -specific inhibitor, PDTC, increased response to a low force (0.16 g) von Frey filament is alleviated in the HbSS mice. n = 6 mice/group. *P<0.05 vs. the baseline in HbAA mice. #P<0.05 vs. the baseline in HbSS mice. (E) Scn10a promoter fragments immunoprecipitated by rabbit anti-p65 in the L3-L5 DRG from HbSS (SS) and HbAA (AA) mice. Top: representative gel image. Bottom: a summary of densitometric analysis. Input: total purified fragments. M: ladder marker. n=3 mice/genotype. **P<0.01 vs. the HbAA mice. (F) Scn10a promoter activity in mouse CAD cells transfected with a luciferase reporter vector containing a Scn10a promoter and treated with drugs as indicated. PMA: the PKC activator. BIM: the PKC-specific inhibitor. PDTC: the NF-κB-specific inhibitor. n=3 repeats/treatment. One-way ANOVA on Ranks, **P<0.01 vs. the vehicle group. #P<0.05 vs. the PMA-treated group. (G) Single cell RT-PCR analysis shows the co-expression of Scn10a mRNA, Ednra mRNA (encoding ET-1), and Rela mRNA (encoding p65) in individual small DRG neurons from HbAA mice. Gapdh mRNA is used as a positive control. M: ladder marker. n=3 repeats.