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    CLS Cell Lines Service GmbH imr 32
    ( A ) Profiling TERRA expression in ALT+ and ALT- NB cell lines (with MYCN amplification [NMA] and without MYCN amplification [Non-NMA]) by slot blot assay performed with 50, 100, and 200 ng of RNA isolated from cell lines indicated. Blot probed with a DIG-labeled TERRA probe. TapeStation profile on the right shows 18s and 28s rRNA served as a loading control. Bar graph shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Data are shown as mean ± SD from three biological replicates. One-way ANOVA with Tukey's post hoc test was used, **** P < 0.0001. ( B ) m 6 A RIP followed by TERRA slot blot on RNA isolated from ALT+ and ALT-cell lines. RIP with IgG severed as a negative control. Bar graph shows the quantification of the blots. Data are shown as mean ± SD from three biological replicates. Sidak's multiple comparisons test was used, **** P < 0.0001; ns - nonsignificant P > 0.05. ( C ) TERRA foci (green) visualized by TERRA RNA-FISH in ALT+ (upper panel), NMA (middle panel) and Non-NMA (lower panel) NB cell lines. Box plot shows the quantification of the TERRA foci per nucleus. At least 67 cells were counted from three independent biological replicates. ( D ) TERRA foci (green) combined with m 6 A (red) IF in CHLA-90 (top) and SK-N-FI (bottom) cells. Box plot shows the number of overlaps of TERRA and m 6 A per nucleus. At least 65 cells were counted from three independent biological replicates. Scale bar is 10 μm. ( E ) The m 6 A enrichment profiles across all subtelomeres using T2T assembly in SK-N-FI and SK-N-BE cells. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. ( F ) Box plot of spike-in, mappability-normalized m 6 A RIP/input enrichment profiles of all subtelomeres in SK-N-FI and SK-N-BE. Statistical significance was calculated using the Wilcoxon test, **** P < 0.0001. ( G , left panel) TERRA expression in ALT+, NMA and Non-NMA NB patient tumor RNA samples were detected by slot blot assay performed with 100 ng of total RNA. (Middle panel) TapeStation profile shows 18s and 28s rRNA served as a loading control, samples are loaded as NB1 to NB13 from top to bottom. (Right panel) Box plot shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Two-way ANOVA with Tukey's post hoc test was used, * P < 0.05; ns - nonsignificant P > 0.05. ( H ) Representative browser screenshot of 50 kb region around the telomere ends, showing TERRA RNA expression (CPM) from two of the active chromosome ends (Chr9p, Chr16q) and one inactive chromosome end (Chr3p) using Illumina short read RNA-seq performed on NB1 and NB2 tumor samples. CpG islands are marked with green bars and the telomeric repeats in this region are marked with black bars. ( I ) Heatmap summarizing RNA-seq and m 6 A RIP-seq data of two NB tumor samples (NB1 and NB2). Chromosomes are sorted based on high (dark-colored) and low (light-colored) subtelomeric TERRA transcription. Black dots denote m 6 A RIP-seq peaks identified using MACS peak caller in these NB tumor samples. ( J ) Genome browser screenshots showing m 6 A RIP/input ratio tracks for NB1 and NB2 tumor samples at two active chromosome ends (Chr12p, Chr20q) and one inactive chromosome end (Chr3p). Red bars mark m 6 A RIP peaks identified using MACS peak caller. ( K ) The m 6 A enrichment profiles across subtelomeres using T2T assembly in NB1 and NB2 tumors. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. Subtelomeres (high and low TERRA transcription) were classified according to the normalized median expression shown. ( L ) Event-free survival of NB patients (n = 498, SEQC cohort) with either low (blue) or high (red) expression of METTL3. ( M ) Event-free survival of ALT+ NB patients (n = 21) with either low (blue) or high (red) expression of METTL3. ( N ) Bar graph showing the percentage of ALT+ or NMA tumors with copy number gain in METTL3 , METTL14 , or hnRNPA2B1 genes. ( O ) Immunohistochemistry (IHC) analysis of METTL3 and METTL14 in <t>human</t> <t>neuroblastoma</t> tumors belonging to either of the subgroups (ALT+, NMA- high risk, or low risk). Sections were counterstained with hematoxylin. One representative image is presented from each subgroup of NB patients.
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    ( A ) Profiling TERRA expression in ALT+ and ALT- NB cell lines (with MYCN amplification [NMA] and without MYCN amplification [Non-NMA]) by slot blot assay performed with 50, 100, and 200 ng of RNA isolated from cell lines indicated. Blot probed with a DIG-labeled TERRA probe. TapeStation profile on the right shows 18s and 28s rRNA served as a loading control. Bar graph shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Data are shown as mean ± SD from three biological replicates. One-way ANOVA with Tukey's post hoc test was used, **** P < 0.0001. ( B ) m 6 A RIP followed by TERRA slot blot on RNA isolated from ALT+ and ALT-cell lines. RIP with IgG severed as a negative control. Bar graph shows the quantification of the blots. Data are shown as mean ± SD from three biological replicates. Sidak's multiple comparisons test was used, **** P < 0.0001; ns - nonsignificant P > 0.05. ( C ) TERRA foci (green) visualized by TERRA RNA-FISH in ALT+ (upper panel), NMA (middle panel) and Non-NMA (lower panel) NB cell lines. Box plot shows the quantification of the TERRA foci per nucleus. At least 67 cells were counted from three independent biological replicates. ( D ) TERRA foci (green) combined with m 6 A (red) IF in CHLA-90 (top) and SK-N-FI (bottom) cells. Box plot shows the number of overlaps of TERRA and m 6 A per nucleus. At least 65 cells were counted from three independent biological replicates. Scale bar is 10 μm. ( E ) The m 6 A enrichment profiles across all subtelomeres using T2T assembly in SK-N-FI and SK-N-BE cells. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. ( F ) Box plot of spike-in, mappability-normalized m 6 A RIP/input enrichment profiles of all subtelomeres in SK-N-FI and SK-N-BE. Statistical significance was calculated using the Wilcoxon test, **** P < 0.0001. ( G , left panel) TERRA expression in ALT+, NMA and Non-NMA NB patient tumor RNA samples were detected by slot blot assay performed with 100 ng of total RNA. (Middle panel) TapeStation profile shows 18s and 28s rRNA served as a loading control, samples are loaded as NB1 to NB13 from top to bottom. (Right panel) Box plot shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Two-way ANOVA with Tukey's post hoc test was used, * P < 0.05; ns - nonsignificant P > 0.05. ( H ) Representative browser screenshot of 50 kb region around the telomere ends, showing TERRA RNA expression (CPM) from two of the active chromosome ends (Chr9p, Chr16q) and one inactive chromosome end (Chr3p) using Illumina short read RNA-seq performed on NB1 and NB2 tumor samples. CpG islands are marked with green bars and the telomeric repeats in this region are marked with black bars. ( I ) Heatmap summarizing RNA-seq and m 6 A RIP-seq data of two NB tumor samples (NB1 and NB2). Chromosomes are sorted based on high (dark-colored) and low (light-colored) subtelomeric TERRA transcription. Black dots denote m 6 A RIP-seq peaks identified using MACS peak caller in these NB tumor samples. ( J ) Genome browser screenshots showing m 6 A RIP/input ratio tracks for NB1 and NB2 tumor samples at two active chromosome ends (Chr12p, Chr20q) and one inactive chromosome end (Chr3p). Red bars mark m 6 A RIP peaks identified using MACS peak caller. ( K ) The m 6 A enrichment profiles across subtelomeres using T2T assembly in NB1 and NB2 tumors. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. Subtelomeres (high and low TERRA transcription) were classified according to the normalized median expression shown. ( L ) Event-free survival of NB patients (n = 498, SEQC cohort) with either low (blue) or high (red) expression of METTL3. ( M ) Event-free survival of ALT+ NB patients (n = 21) with either low (blue) or high (red) expression of METTL3. ( N ) Bar graph showing the percentage of ALT+ or NMA tumors with copy number gain in METTL3 , METTL14 , or hnRNPA2B1 genes. ( O ) Immunohistochemistry (IHC) analysis of METTL3 and METTL14 in human neuroblastoma tumors belonging to either of the subgroups (ALT+, NMA- high risk, or low risk). Sections were counterstained with hematoxylin. One representative image is presented from each subgroup of NB patients.

    Journal: Nucleic Acids Research

    Article Title: METTL3 drives telomere targeting of TERRA lncRNA through m 6 A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma

    doi: 10.1093/nar/gkad1242

    Figure Lengend Snippet: ( A ) Profiling TERRA expression in ALT+ and ALT- NB cell lines (with MYCN amplification [NMA] and without MYCN amplification [Non-NMA]) by slot blot assay performed with 50, 100, and 200 ng of RNA isolated from cell lines indicated. Blot probed with a DIG-labeled TERRA probe. TapeStation profile on the right shows 18s and 28s rRNA served as a loading control. Bar graph shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Data are shown as mean ± SD from three biological replicates. One-way ANOVA with Tukey's post hoc test was used, **** P < 0.0001. ( B ) m 6 A RIP followed by TERRA slot blot on RNA isolated from ALT+ and ALT-cell lines. RIP with IgG severed as a negative control. Bar graph shows the quantification of the blots. Data are shown as mean ± SD from three biological replicates. Sidak's multiple comparisons test was used, **** P < 0.0001; ns - nonsignificant P > 0.05. ( C ) TERRA foci (green) visualized by TERRA RNA-FISH in ALT+ (upper panel), NMA (middle panel) and Non-NMA (lower panel) NB cell lines. Box plot shows the quantification of the TERRA foci per nucleus. At least 67 cells were counted from three independent biological replicates. ( D ) TERRA foci (green) combined with m 6 A (red) IF in CHLA-90 (top) and SK-N-FI (bottom) cells. Box plot shows the number of overlaps of TERRA and m 6 A per nucleus. At least 65 cells were counted from three independent biological replicates. Scale bar is 10 μm. ( E ) The m 6 A enrichment profiles across all subtelomeres using T2T assembly in SK-N-FI and SK-N-BE cells. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. ( F ) Box plot of spike-in, mappability-normalized m 6 A RIP/input enrichment profiles of all subtelomeres in SK-N-FI and SK-N-BE. Statistical significance was calculated using the Wilcoxon test, **** P < 0.0001. ( G , left panel) TERRA expression in ALT+, NMA and Non-NMA NB patient tumor RNA samples were detected by slot blot assay performed with 100 ng of total RNA. (Middle panel) TapeStation profile shows 18s and 28s rRNA served as a loading control, samples are loaded as NB1 to NB13 from top to bottom. (Right panel) Box plot shows the TERRA level quantified from blots and normalized to 28s rRNA loading control. Two-way ANOVA with Tukey's post hoc test was used, * P < 0.05; ns - nonsignificant P > 0.05. ( H ) Representative browser screenshot of 50 kb region around the telomere ends, showing TERRA RNA expression (CPM) from two of the active chromosome ends (Chr9p, Chr16q) and one inactive chromosome end (Chr3p) using Illumina short read RNA-seq performed on NB1 and NB2 tumor samples. CpG islands are marked with green bars and the telomeric repeats in this region are marked with black bars. ( I ) Heatmap summarizing RNA-seq and m 6 A RIP-seq data of two NB tumor samples (NB1 and NB2). Chromosomes are sorted based on high (dark-colored) and low (light-colored) subtelomeric TERRA transcription. Black dots denote m 6 A RIP-seq peaks identified using MACS peak caller in these NB tumor samples. ( J ) Genome browser screenshots showing m 6 A RIP/input ratio tracks for NB1 and NB2 tumor samples at two active chromosome ends (Chr12p, Chr20q) and one inactive chromosome end (Chr3p). Red bars mark m 6 A RIP peaks identified using MACS peak caller. ( K ) The m 6 A enrichment profiles across subtelomeres using T2T assembly in NB1 and NB2 tumors. The m 6 A RIP/input signals were normalized to spike-in and to the mappability likelihood of each chromosome ends. Subtelomeres (high and low TERRA transcription) were classified according to the normalized median expression shown. ( L ) Event-free survival of NB patients (n = 498, SEQC cohort) with either low (blue) or high (red) expression of METTL3. ( M ) Event-free survival of ALT+ NB patients (n = 21) with either low (blue) or high (red) expression of METTL3. ( N ) Bar graph showing the percentage of ALT+ or NMA tumors with copy number gain in METTL3 , METTL14 , or hnRNPA2B1 genes. ( O ) Immunohistochemistry (IHC) analysis of METTL3 and METTL14 in human neuroblastoma tumors belonging to either of the subgroups (ALT+, NMA- high risk, or low risk). Sections were counterstained with hematoxylin. One representative image is presented from each subgroup of NB patients.

    Article Snippet: NB cell lines SK-N-FI (Sigma-Aldrich), SK-N-AS (ATCC), SH-SY5Y (CLS Cell Lines Service), CHLA-90 (CCOG), IMR-32 (CLS Cell Lines Service) and SK-N-BE(2) (DSMZ) cells were cultured in specific media.

    Techniques: Expressing, Amplification, Slot Blot Assay, Isolation, Labeling, Dot Blot, Negative Control, RNA Expression, RNA Sequencing Assay, Immunohistochemistry