Figure 1
Knockdown of MCT1 in primary cultures of hypothalamic tanycytes.
(A) Experimental protocol that shows the construction of adenoviral shuttle vector. The cassette encoding the H1-promotor, multicloning site (MCS), ubiquitin promoter, EGFP and SV40 polyA contained in the Fux vector was cloned into the PDC311 adenoviral shuttle expression vector for generating the vector, PDC311.2. The shRNAs were cloned into the MCS. (B–G) Temporal EGFP expression in tanycytes cultures transduced for 48, 72 and 96 h with AdshβGal (B–D) or AdshMCT1 (E–G). Nuclei were stained with TOPRO-3 (blue). (H) Quantification of EGFP expression normalized to total cells in the tanycyte cultures transduced with AdshβGal (open bars) or AdshMCT1 (closed bars) at 48, 72 and 96 h. (I) Quantification of survival of tanycytes cultures transduced with AdshMCT1 at 48, 72 and 96 h relative to survival of cells transduced with AdshβGal. (J) Q-RT-PCR analysis of MCT1 in tanycytes infected with AdshβGal (open bars) or AdshMCT1 (closed bars) at 48, 72 and 96 h. (K) Western blot analysis of MCT1, GFP and actin. Lanes 1–3: Total cell extracts treated for 96 h with AdshβGal; lanes 4–6: total cell extracts treated for 96 h with AdshMCT1. (L) Semi-quantitative densitometric analysis of MCT1 protein expression relative to actin. GFP: Transduction control. Actin: Loading control. **p < 0.01 (unpaired t-test). Scale bar: 25 μm. MCS, Multicloning site; pH1, H1 promoter; pUb, ubiquitin promoter; SV40-poly A, polyadenylation sequence from Simian virus 40; T4, DNA ligase from bacteriophage, T4.
