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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml <t>RNase</t> <t>A</t> or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.
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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml <t>RNase</t> <t>A</t> or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.
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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml <t>RNase</t> <t>A</t> or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.
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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml <t>RNase</t> <t>A</t> or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.
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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml <t>RNase</t> <t>A</t> or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.
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GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml RNase A or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.

Journal: Nucleic Acids Research

Article Title: AquIRE reveals the mechanisms of clinically induced RNA damage and the conservation and dynamics of glycoRNAs

doi: 10.1093/nar/gkag080

Figure Lengend Snippet: GlycoRNAs are elevated by and determine response to RNA-damaging chemotherapy. ( A ) Schematic of colorectal tumour processing to generate unsorted cell pellets and cell-free tumour material. RNA was extracted from each fraction for analysis. Figure generated in BioRender: https://BioRender.com/s88×264 . ( B ) RNA from panel (A) was analysed for glycoRNA PNA by AquIRE and plotted as the fluorescence per µg of RNA for five biologically independent tumours, showing the mean of at least two technical replicates. The x- axis lists the unique tumour name. ( C ) HCT116 colorectal cancer cells were treated with 10 µM 5FU for 72 h, 50 µM oxaliplatin for 6 h, or their vehicles, and U251 glioblastoma cells were treated with 2 mM temozolomide or vehicle for 2 h. GlycoRNA PNA content was determined in RNA extracted after these treatments, with the graphs plotting the mean fluorescence per µg from three biological replicates per drug ± SEM. Significance was determined by unpaired t- test. ( D ) HCT116 cells were treated with 10 µM 5FU for 72 h in the presence of 100 or 200 µg/ml RNase A or no enzyme. Right, the change in Cell Titer Glo viability analysis signal was plotted relative to vehicle with no enzyme set to 100%. Due to variability in the efficacy of 5FU between biological replicates (5% SEM) values after drug treatment were normalized against the average effect of 5FU in the absence of enzyme (49.3%). Right, the ratio of the cell viability signal of 5FU/vehicle for the three different enzyme conditions was plotted as a percentage change in viability compared to no-enzyme set to 0. Data represent the mean of three biological replicates ± SEM. Significance compared to no-enzyme condition was tested using an ANOVA with Šídák multiple-comparison testing. * P < .05.

Article Snippet: RNase A digests : Cells were treated with non-cell permeable RNase A (NEB) concurrently with drug treatments by diluting the enzyme directly in cell media.

Techniques: Generated, Fluorescence, Comparison