Journal: PLoS Pathogens
Article Title: miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila
Figure Lengend Snippet: Over-expression of miR-34 activates innate immunity signaling. ( A ) Total RNA was isolated from male progeny from crossing flies carrying the ubiquitously expressed da-Gal4 driver and a temperature-sensitive Gal80 transgene ( da > Gal4 tub-Gal80 ts ) to UAS-shRNA lines targeting Drosha or the control gfp . Flies crosses were kept at 18°C and progeny were shifted to 29°C for 5 days upon eclosure to induce shRNA expression. Steady-state levels of mRNAs encoding Drosha and several primary miRNA transcripts were measured by qRT-PCR, and normalized to levels of the RpL32 mRNA. RNA isolated from da > gfp shRNA males serves as negative control. (n = 3). ( B ) Flies were left untreated (- E . coli ) or infected by E . coli via septic injury (+ E . coli ), total RNAs were extracted 6 hours post-infection and mRNAs encoding the AMP Diptericin was measured and normalized to levels of RpL32 (n = 5; mean + standard deviation ( SD )). ( C-E ) Select miRNAs were over-expressed in flies by crossing UAS-miRNA transgenic lines da > Gal4 tub-Gal80 ts flies. Flies crosses were kept at 18°C and progeny were shifted to 29°C for 5 days upon eclosure to induce miRNA expression. ( C ) Northern blot shows levels of select miRNAs (right) in control and miRNA over-expression flies. 2S rRNA serves as loading control. In addition, flies were either uninfected ( D ) or infected with E . coli via septic injury ( E ). Total RNA was isolated from flies 6 hrs post-infection and levels of Diptericin mRNA were measured by RT-qPCR and normalized to the RpL32 mRNA. RNA samples from da > gfp shRNA flies serves as control. Note that levels of the Diptericin mRNA in non-infected and E . coli -infected da > gfp shRNA flies serve as baseline controls in both D and E (n≥4). ( F ) A Northern blot shows miR-34 expression levels in naïve S2 cells and miR-34 overexpression cells (both were treated with 20-HE at 1 μM for 24 hrs). ( G ) S2 cells over-expressing miR-34 and control cells were both treated with 20 hydroxy-ecdysone ( 20-HE ) at 1 μM for 24 hrs. Subsequently cells were either left untreated or treated for 6 hrs with a crude lipopolysaccharide sample at 10 μg/mL, which contains the immune stimulator p eptido g lyca n ( PGN ). Total RNA was isolated and levels of Diptericin mRNA were measured by RT-qPCR and normalized to the RpL32 mRNA (n = 3). ( H ) Canonical components of IMD signaling were depleted in miR-34 over-expressing cells using dsRNAs targeting IMD pathway components (below) or a control dsRNA against the firefly luciferase gene. Cells were first treated with 20-HE for 24 hours, and subsequently were either left untreated or treated with PGN, and levels of Diptericin mRNA were measured by RT-qPCR and normalized to the RpL32 mRNA (n = 3). ( I ) UAS-miR-34 or the control UAS-sh-gfp flies were crossed to da > Gal4 tub-Gal80 ts flies. Flies crosses were kept at 18°C. Upon eclosure, progeny of appropriate genotypes were collected and shifted to 29°C for 7 days. Flies in groups of 45 were subsequently injected with a concentrated culture of Erwinia carotovora carotovora 15 ( Ecc15 ) or PBS (non-infection control) and kept at 29°C. Fly survival was recorded daily up to day 8 post-infection and plotted (n≥3; p
Article Snippet: Subsequently cells were treated with 10 μg/mL crude LPS prep (Sigma), which contains peptidoglycan (PGN) as a potent inducer of IMD signaling [ ], for another 6 hours.
Techniques: Over Expression, Isolation, shRNA, Expressing, Quantitative RT-PCR, Negative Control, Infection, Standard Deviation, Transgenic Assay, Northern Blot, Radial Immuno Diffusion, Luciferase, Injection