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. 2018 May 14;128(6):2626–2641. doi: 10.1172/JCI99350

Figure 5. Silencing of galanin in the VHT of AgRP-CRE mice prevents ΔFosB-induced elevation of energy metabolism and bone density in AgRP neurons.

Figure 5

(A) CRE-inducible FLAG-tagged ΔFosB pTomo or empty pTomo constructs were modified to incorporate the H1 promoter and scrambled or galanin shRNA. These constructs were packaged into LVs and stereotactically delivered to VHT of 6- to 7-week-old male neuronal promoter AgRP-CRE mice for simultaneous ΔFosB expression and galanin silencing in selected neurons. Energy metabolism and bone were analyzed 6 weeks after surgery. (B) Real-time PCR and Western blot validation of galanin mRNA and protein suppression in the mHypoE 42 hypothalamic cell line transfected and selected with G418 for stable expression of modified constructs (n = 4). (C) Western blot validation of ΔFosB protein overexpression in the presence of CRE using the mHypoE 42 cell line transfected with modified constructs (n = 3). (D) Calorimetric analysis of metabolism. (E) Micro-CT analysis of femurs (see Table 2 for values). (F) Histomorphometric analysis of tibiae. (G) Serum P1NP and CTX. Statistical analysis included ANOVA followed by Tukey-Kramer HSD test comparing 4 groups (n = 6–9), *P < 0.05. Groups marked by different letters significantly differ from each other (e.g., a is significantly different from b, but a is not significantly different from ab).