Figure 3.

Intratracheal (IT) overexpression of Rtp801 suppresses pulmonary mTORC1 activity and augments LPS-induced lung inflammation. A: Intratracheal transfection of Tg-TRE–RTP-801 mice with the tet-off TTA adenovirus increases pulmonary Rtp801 expression by 91% in comparison to Tg-TRE–RTP-801 mice transfected with a GFP-expressing adenoviral control. Expression is quantified as 2^−ΔΔC_T, normalized to both the housekeeping gene cyclophilin A and GFP-AAV–treated controls. B: Confirmation of Rtp801 (red) overexpression within alveoli of TTA-AAV–pretreated, LPS-treated Tg-TRE–RTP-801 mice using immunofluorescence. C: Transgenic Rtp801 overexpression appropriately functions to suppress mTORC1, as demonstrated by a dramatic attenuation of S6 phosphorylation (p-S6) 4 hours after 2 mg/kg intratracheal LPS. High-magnification inset demonstrates p-S6 colocalization (arrows) with SPC. DAPI = nuclear stain. D: Rtp801 overexpression augments pulmonary neutrophilic inflammation, as apparent in BAL 24 hours after 2 mg/kg intratracheal LPS. E: These findings are supported by a trend toward increased lung injury in LPS-treated, Rtp801-overexpressing mice. ^∗P < 0.05, ^†P = 0.05. n = 3 to 4 per group. Scale bars: 50 μm (B and C); 100 μm (E).