Figure 5. Omega-1 suppresses protein... | Rockefeller University Press

Figure 5.

$Figure 5. Omega-1 suppresses protein synthesis through breakdown of rRNA and mRNA. (A) After human moDCs had been pulsed for 40 h with 125, 250, and 500 ng/ml omega-1 (ω-1) in combination with 100 ng/ml LPS, the cells were co-cultured for 24 h with the J558 cell line, expressing CD40-L, to mimic the interaction with T cells. Bars represent mean ± SD of triplicate wells of one of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for values significantly different from the LPS control. (B) After16-h incubation of human DCs with a concentration range of indicated reagents in the presence of 100 ng/ml LPS, protein synthesis was assessed after a 2-h pulse with radioactively labeled methionine. Ricin, as potent inhibitor of protein synthesis, was taken along as positive control (Montanaro et al., 1973). One of two experiments is shown. Data points represent mean ± SD of duplicates. (C) As described in B, protein synthesis by human DCs was followed over time after exposure to 500 ng/ml omega-1 either in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls as depicted by the dotted line. Data are representative of two independent experiments and are depicted as mean ± SD. (D) Protein synthesis by human moDCs after exposure to increasing concentrations of the recombinant omega-1 variants was assessed as described in B. Data are shown relative to LPS-stimulated DCs. Data are representative of two independent experiments and are depicted as mean ± SD. (E) DCs were stimulated with FITC-labeled recombinant omega-1 for 1 h, and uptake was visualized by confocal laser-scanning microscopy. Nuclei were stained with Hoechst. One of three experiments is shown. See Video 1 for z-stacked images. (F) Cytoplasmic fractions of human DCs stimulated for 3 h with omega-1 were run under nonreducing conditions by SDS-PAGE and analyzed by Western blot for the presence of omega-1 or silver stained to control for input. DCs incubated at 4°C were taken along as controls as these cells have surface-bound but not internalized omega-1. One of two experiments is shown. (G) Human DCs were stimulated with 1 µg/ml FITC-labeled omega-1 and after 2 h fixed and stained for rRNA. Subcellular localization of omega-1 was determined by confocal microscopy. One representative cell from three independent experiments is shown. Arrowheads depict areas where rRNA and omega-1 colocalize (yellow). Bars, 10 µm. (H) After rabbit reticulocyte lysate containing functional ribosomes was incubated for 1 h with 1 and 5 µg/ml omega-1, 1 and 5 µg/ml IPSE as negative control, or 25 µg/ml ES (schistosome egg excretory/secretory products), containing omega-1, isolated rRNA was analyzed for breakdown on a 2% agarose gel. The RNase α-sarcin was taken along as positive control as it should give a single rRNA cleavage product when incubated with functional ribosomes (arrowhead; Kao et al., 2001). One of three independent experiments is shown. (I) rRNA was isolated from 24-h omega-1–stimulated human DCs and was visualized by running a laboratory-on-a-chip picogel. One of three experiments is shown. (J and K) rRNA or mRNA expression of the indicated genes in DCs was assessed by real-time quantitative PCR at different time points after stimulation with 500 ng/ml and 2 µg/ml omega-1 in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls, which were set to 1. RNA expression was normalized based on a genomic real-time quantitative PCR for ccr5. Data represent the mean of three independent experiments. H58F, RNase mutant; N71/176Q, glycosylation mutant.$

Omega-1 suppresses protein synthesis through breakdown of rRNA and mRNA. (A) After human moDCs had been pulsed for 40 h with 125, 250, and 500 ng/ml omega-1 (ω-1) in combination with 100 ng/ml LPS, the cells were co-cultured for 24 h with the J558 cell line, expressing CD40-L, to mimic the interaction with T cells. Bars represent mean ± SD of triplicate wells of one of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for values significantly different from the LPS control. (B) After16-h incubation of human DCs with a concentration range of indicated reagents in the presence of 100 ng/ml LPS, protein synthesis was assessed after a 2-h pulse with radioactively labeled methionine. Ricin, as potent inhibitor of protein synthesis, was taken along as positive control (Montanaro et al., 1973). One of two experiments is shown. Data points represent mean ± SD of duplicates. (C) As described in B, protein synthesis by human DCs was followed over time after exposure to 500 ng/ml omega-1 either in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls as depicted by the dotted line. Data are representative of two independent experiments and are depicted as mean ± SD. (D) Protein synthesis by human moDCs after exposure to increasing concentrations of the recombinant omega-1 variants was assessed as described in B. Data are shown relative to LPS-stimulated DCs. Data are representative of two independent experiments and are depicted as mean ± SD. (E) DCs were stimulated with FITC-labeled recombinant omega-1 for 1 h, and uptake was visualized by confocal laser-scanning microscopy. Nuclei were stained with Hoechst. One of three experiments is shown. See Video 1 for z-stacked images. (F) Cytoplasmic fractions of human DCs stimulated for 3 h with omega-1 were run under nonreducing conditions by SDS-PAGE and analyzed by Western blot for the presence of omega-1 or silver stained to control for input. DCs incubated at 4°C were taken along as controls as these cells have surface-bound but not internalized omega-1. One of two experiments is shown. (G) Human DCs were stimulated with 1 µg/ml FITC-labeled omega-1 and after 2 h fixed and stained for rRNA. Subcellular localization of omega-1 was determined by confocal microscopy. One representative cell from three independent experiments is shown. Arrowheads depict areas where rRNA and omega-1 colocalize (yellow). Bars, 10 µm. (H) After rabbit reticulocyte lysate containing functional ribosomes was incubated for 1 h with 1 and 5 µg/ml omega-1, 1 and 5 µg/ml IPSE as negative control, or 25 µg/ml ES (schistosome egg excretory/secretory products), containing omega-1, isolated rRNA was analyzed for breakdown on a 2% agarose gel. The RNase α-sarcin was taken along as positive control as it should give a single rRNA cleavage product when incubated with functional ribosomes (arrowhead; Kao et al., 2001). One of three independent experiments is shown. (I) rRNA was isolated from 24-h omega-1–stimulated human DCs and was visualized by running a laboratory-on-a-chip picogel. One of three experiments is shown. (J and K) rRNA or mRNA expression of the indicated genes in DCs was assessed by real-time quantitative PCR at different time points after stimulation with 500 ng/ml and 2 µg/ml omega-1 in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls, which were set to 1. RNA expression was normalized based on a genomic real-time quantitative PCR for ccr5. Data represent the mean of three independent experiments. H58F, RNase mutant; N71/176Q, glycosylation mutant.

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