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Results: 5

1.
Figure 1

Figure 1. Production of fluorescent GeRPs. From: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

β1,3-d-glucan particles were purified from baker’s yeast by a series of alkaline and solvent extractions, hydrolysing outer cell wall and intracellular components and yielding purified, porous 2–4 µm, hollow β1,3-d-glucan particles (diagram of particles, left; procedure, middle; microscopy of particles, right). Empty β1,3-d-glucan particles were then labelled with fluorescein (FL, green). The cores were synthesized by absorbing yeast tRNA, PEI and Dy547-labelled siRNA (red) in a layer-by-layer format. Bottom right: confocal image of a synthesized GeRP.

Myriam Aouadi, et al. Nature. ;458(7242):1180-1184.
2.
Figure 3

Figure 3. Map4k4 silencing fails to affect LPS activation of JNK, p38 and NFκB signalling pathways. From: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

PECs treated with GeRPs containing scrambled (Scr) or Map4k4 (oligo 1) siRNA, and stimulated with 1 µg ml−1 LPS for the indicated amounts of time were analysed for phospho- (P-) and total JNK1/2 (a) and p38 MAPK (b), and for total IκBα (c). Representative blots are shown from three experiments. Immunoblot signals are expressed in arbitrary units (a.u.) and data represent mean ± s.e.m. (n = 3). Filled squares, Scr siRNA; open triangles, Map4k4 siRNA. d, Schematic diagram of potential Map4k4 signalling to modulate the expression of inflammatory genes such as Tnf and Il1b.

Myriam Aouadi, et al. Nature. ;458(7242):1180-1184.
3.
Figure 4

Figure 4. Orally administered GeRPs containing Map4k4 siRNA attenuate Map4k4 mRNA expression in PECs and macrophages from spleen, lung and liver. From: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

a, Time line of GeRP (scrambled (Scr) or Map4k4, oligo 1) administration and PEC/tissue isolation. b, Confocal microscopy of PECs and tissue macrophages containing GeRPs (green). Macrophages were stained with F4/80–AF633 (magenta). Arrows indicate cells containing GeRPs. Nuclei (n) were stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Magnification is indicated in the panels. c, Map4k4 mRNA expression in PECs and adherent cells from tissues. d, Serum INF-γ levels from mice gavaged with PBS, unloaded GeRPs or GeRPs containing 20 µg kg−1 of Scr siRNA (n = 5). Statistical significance was determined by ANOVA and Tukey post test. **P < 0.01 and *P < 0.05. Results are mean ± s.e.m.

Myriam Aouadi, et al. Nature. ;458(7242):1180-1184.
4.
Figure 5

Figure 5. Map4k4 silencing by oral gavage with GeRPs inhibits LPS-induced Tnf-α production in vivo. From: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

a, Time line of siRNA and LPS/d-GalN administration. b, Tnf, Il1b, Il10 and Ccr2 mRNA expression in unstimulated PECs from mice gavaged with Scr- and Map4k4-siRNA-containing GeRPs (n = 3). Statistical significance was determined by a two-tailed Student’s t-test. c, d, Serum (c) and peritoneal (d) Tnf-α levels in siRNA-treated mice 1.5 h and 4 h after LPS/d-GalN administation (n = 10). Statistical significance was determined by ANOVA and Tukey post test; **P < 0.001 and *P < 0.05. e, Survival percentage of mice gavaged with PBS or Scr- and Map4k4-siRNA-containing GeRPs, followed by LPS/d-GalN administration (PBS, n = 11; Scr and Map4k4, n = 22). Statistical significance was determined by Kaplan–Meier analysis and Mantel–Cox testing (P < 0.01; see Supplementary Table 3). Results are the mean of three independent experiments. All results are mean ± s.e.m.

Myriam Aouadi, et al. Nature. ;458(7242):1180-1184.
5.
Figure 2

Figure 2. In vitro treatment of GeRPs containing Map4k4 siRNA silence Map4k4 expression and inhibit LPS-induced Tnf-α production in macrophages. From: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

a, Confocal images of cultured PECs treated with siRNA–GeRPs (red/green). PECs were stained for F4/80 (blue) and nuclei (n) (Hoescht; blue). Arrows indicate GeRPs. b, Map4k4 mRNA expression from PECs treated with increasing Map4k4 siRNA concentrations. a.u., arbitrary units. c, Map4k4 and Tnf mRNA expression from PECs treated with PBS, unloaded GeRPs or scrambled (Scr) GeRPs. d, Tnf mRNA expression from PECs treated with Scr- or Map4k4-siRNA-containing GeRPs, ±6 h LPS (1 µg ml−1). e, Tnf-α secretion from PECs treated with Scr or Map4k4 GeRPs. Statistical significance was determined by ANOVA and Tukey post test. *P < 0.05. Results are mean ± s.e.m. f, PCR amplification of 5′ RACE products produces a band (arrow) reflecting the predicted RNAi-mediated mRNA cleavage product in Map4k4- but not Scr-siRNA-treated PECs.

Myriam Aouadi, et al. Nature. ;458(7242):1180-1184.

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