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a172  (ATCC)


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    ATCC a172
    Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the <t>A172,</t> LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P < 0.01
    A172, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 2093 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/genomics+suite+(software+analysis+microarray+data/A-172/pmc06851045-17-6-13
    Average 97 stars, based on 2093 article reviews
    a172 - by Bioz Stars, 2026-08
    97/100 stars

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    1) Product Images from "Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program"

    Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-019-02062-4

    Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the A172, LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P < 0.01
    Figure Legend Snippet: Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the A172, LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P < 0.01

    Techniques Used: Methylation, Expressing, Genomic Sequencing, Clone Assay, DNA Methylation Assay, Microarray, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

    Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d
    Figure Legend Snippet: Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d

    Techniques Used: In Vivo, Western Blot, Expressing, Stable Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay, Injection

    NSUN5 epigenetic inactivation abrogates the methylation of the C3782 position of human 28S rRNA. a Top , RNA sequence alignment of the conserved human 28S rRNA C3782 position (black square) in the corresponding 26S, 25S and 28S rRNAs orthologues from C. elegans , S. cerevisiae and M. musculus . Below , Protein sequence alignment of human NSUN5 with its recognized rRNA 5-methylcytosine RNA-methyltransferase orthologues in C. Elegans , S. Cerevisiae and M. musculus . Highlighted in black and grey the identical and physicochemically similar (scoring > 0.5 in the Gonnet PAM 250 matrix) residues, respectively. The aligned region includes the RNA-methyltransferase domain. b NSUN5 interaction with 28S rRNA. Total extracts from LN229 cells, either transfected with empty vector (EV) or NSUN5 were immunoprecipitated with an anti-Flag antibody ( left panel ), followed by analysis of the retrieved RNA by quantitative RT-PCR ( right panel ). c RNA bisulfite sequencing of the 28S rRNA in glioma cells lines according to NSUN5 epigenetic status. Cytosines are represented as short vertical lines and the C3782 site is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. d RNA bisulfite sequencing of the 28S rRNA in empty-vector (EV) and NSUN5-transfected LN229 and A172 glioma cells. e RNA bisulfite sequencing of the 28S rRNA in scramble and NSUN5-shRNA-depleted DBTRG-05MG and CAS-1 glioma cells. For CAS-1, western-blot to show efficient NSUN5 depletion is shown above
    Figure Legend Snippet: NSUN5 epigenetic inactivation abrogates the methylation of the C3782 position of human 28S rRNA. a Top , RNA sequence alignment of the conserved human 28S rRNA C3782 position (black square) in the corresponding 26S, 25S and 28S rRNAs orthologues from C. elegans , S. cerevisiae and M. musculus . Below , Protein sequence alignment of human NSUN5 with its recognized rRNA 5-methylcytosine RNA-methyltransferase orthologues in C. Elegans , S. Cerevisiae and M. musculus . Highlighted in black and grey the identical and physicochemically similar (scoring > 0.5 in the Gonnet PAM 250 matrix) residues, respectively. The aligned region includes the RNA-methyltransferase domain. b NSUN5 interaction with 28S rRNA. Total extracts from LN229 cells, either transfected with empty vector (EV) or NSUN5 were immunoprecipitated with an anti-Flag antibody ( left panel ), followed by analysis of the retrieved RNA by quantitative RT-PCR ( right panel ). c RNA bisulfite sequencing of the 28S rRNA in glioma cells lines according to NSUN5 epigenetic status. Cytosines are represented as short vertical lines and the C3782 site is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. d RNA bisulfite sequencing of the 28S rRNA in empty-vector (EV) and NSUN5-transfected LN229 and A172 glioma cells. e RNA bisulfite sequencing of the 28S rRNA in scramble and NSUN5-shRNA-depleted DBTRG-05MG and CAS-1 glioma cells. For CAS-1, western-blot to show efficient NSUN5 depletion is shown above

    Techniques Used: Methylation, Sequencing, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, shRNA, Western Blot

    NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value < 0.05)
    Figure Legend Snippet: NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value < 0.05)

    Techniques Used: Methylation, Transfection, Comparison, RNA Sequencing, Sequencing, Plasmid Preparation

    NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P < 0.0001). e IC50 determination using the SRB assay in NSUN5-transfected LN299 cells in comparison with empty-vector-transfected cells (EV) shows enhanced resistance to DNQ-mediated growth inhibition in the cells with restored NSUN5 expression. Comparison of the IC50 values calculated from the slopes was obtained by means of a z test ( P < 0.001). f Kaplan–Meier analysis of Survival according treatment conditions (IB-DNQ treated vs mock group) in a set of mice models with implanted tumors derived from the NSUN5 methylated cell line A172 (left) or the NSUN5 unmethylated cell line DBTRG (right). Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the hazards ratio (HR) and 95% confidence interval (CI)
    Figure Legend Snippet: NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P < 0.0001). e IC50 determination using the SRB assay in NSUN5-transfected LN299 cells in comparison with empty-vector-transfected cells (EV) shows enhanced resistance to DNQ-mediated growth inhibition in the cells with restored NSUN5 expression. Comparison of the IC50 values calculated from the slopes was obtained by means of a z test ( P < 0.001). f Kaplan–Meier analysis of Survival according treatment conditions (IB-DNQ treated vs mock group) in a set of mice models with implanted tumors derived from the NSUN5 methylated cell line A172 (left) or the NSUN5 unmethylated cell line DBTRG (right). Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the hazards ratio (HR) and 95% confidence interval (CI)

    Techniques Used: Inhibition, Biomarker Discovery, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Comparison, Western Blot, Methylation, Sulforhodamine B Assay, Over Expression, Generated, Software, Derivative Assay



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