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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer <t>chromatography</t> <t>(2D-TLC).</t> Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.
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Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer chromatography (2D-TLC). Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.

Journal: Genes

Article Title: 5-Fluorouracil Treatment Alters the Efficiency of Translational Recoding

doi: 10.3390/genes8110295

Figure Lengend Snippet: Ribosomal RNA (rRNA) pseudouridine levels as well as rRNA levels in SW-480 cells treated with uracil or 5-FU. ( A ) 18S and 28S rRNA were gel-purified from [ 32 P]-orthophosphate pulsed SW-480 cells cultured in medium containing uracil (left panel) or 5-FU (right panel), and subjected to nuclease P1 digestion and 2-dimensional thin layer chromatography (2D-TLC). Spots corresponding to adenosine (pA), cytosine (pC), guanosine (pG), uridine (pU), and pseudouridine (pΨ) are labeled. For uracil-treated cells (left panel), the pU/pΨ ratio is ~5%; for 5-FU-treated cell (right), the pU/pΨ ratio is ~1%. ( B ) Total RNA from 5-FU- and uracil-treated cells was resolved by electrophoresis, and photographed (top). As a loading control, U2 small nuclear RNA (snRNA) was detected by Northern blotting (bottom). ( C ) Relative levels of 18S and 28S from 5-FU- and uracil-treated cells were quantified.

Article Snippet: 3 μL of the digestion reactions were spotted on to cellulose thin layer chromatography (TLC) plates (Cellulose CEL 400-10, 20 × 20 cm; Macherey-Nagel, Düren, Germany).

Techniques: Purification, Cell Culture, Thin Layer Chromatography, Labeling, Electrophoresis, Control, Northern Blot