imr 32  (CLS Cell Lines Service GmbH)


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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.
    Imr 32, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    Images

    1) Product Images from "METTL3 drives telomere targeting of TERRA lncRNA through m 6 A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkad1242

    ( 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.
    Figure Legend 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.

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

    imr 32  (CLS Cell Lines Service GmbH)


    Bioz Verified Symbol CLS Cell Lines Service GmbH is a verified supplier
    Bioz Manufacturer Symbol CLS Cell Lines Service GmbH manufactures this product  
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    Structured Review

    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.
    Imr 32, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/imr 32/product/CLS Cell Lines Service GmbH
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    imr 32 - by Bioz Stars, 2024-05
    94/100 stars

    Images

    1) Product Images from "METTL3 drives telomere targeting of TERRA lncRNA through m 6 A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkad1242

    ( 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.
    Figure Legend 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.

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

    imr 32  (CLS Cell Lines Service GmbH)


    Bioz Verified Symbol CLS Cell Lines Service GmbH is a verified supplier
    Bioz Manufacturer Symbol CLS Cell Lines Service GmbH manufactures this product  
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  • 94

    Structured Review

    CLS Cell Lines Service GmbH imr 32
    A, (Left panel) The total number of m 6 A peaks identified and (middle panel) identified motifs from de novo motif analysis of m 6 A peaks enriched in MYCN-amplified NB tumor samples. (Right panel) Metagene analysis showing relative m 6 A peak density at genes in both MYCN-amplified NB tumor samples. B, (Left panel) Immunoblot showing METTL3 expression in Dox induced (48 hours) control and METTL3 KD SK-N-BE and <t>IMR-32</t> cells . GAPDH was used as a loading control. The values below indicate the fold change in levels of METTL3. (Right panel) Bar plots show cell viability of SK-N-BE and IMR-32 cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. C, (Left panel) Representative immunoblot showing simultaneous MYCN overexpression and METTL3 KD via Dox induction in SHEP (SHEP MYCN ) cells. GAPDH and vinculin were loading controls. The values below indicate the fold change in levels of METTL3 and MYCN. (Right panel) Bar plot shows the cell viability of SHEP MYCN cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. D, Representative immunoblot showing METTL3 and METTL14 expression following siRNA-mediated METTL3 KD. Vinculin was used as a loading control. The values below indicate the fold change in levels of METTL3 and METTL14. E, Representative images from colony formation assay performed in SK-N-BE cells with METTL3 KD (14 days post Dox induction). F, Top enriched terms associated with DEGs (shCtrl Vs. METTL3 KD) having m 6 A peaks (m 6 A+) in SK-N-BE cells. G, (Left panel) Brightfield images of SK-N-BE cells with stable overexpression of HOXC8 and HOXC9. (Right panel) Representative IF showing TUBB3 (green) and overexpression of MYC-tagged HOXC8 and HOXC9 (red) in SK-N-BE cells. H, (Left panel) Venn diagram comparison of HOXC9 target genes [genes with HOXC9 ChIP-seq peak and 1.5 fold change in expression between control vs HOXC9 overexpression] and differentially expressed between DEGs (shCtrl Vs. METTL3 KD) in SK-N-BE cells. (Right panel) Top enriched terms associated with the overlapping genes from the left panel. I, (Left panel) Interaction network of HOXC8 obtained using STRING-db with default parameters. (Right panel) Proximity ligation assay (PLA) showing the HOXC8 and HOXC9 PLA signal (green) between in SK-N-BE cell nucleus (marked by DAPI). The Negative control shows PLA with only the HOXC8 antibody. Scale bar represents 50 μm. J, (Left panel) Representative IF showing staining for PRPH and MYC-tagged HOXC8 and HOXC9 in SK-N-BE cell transiently overexpressing HOXC8 and HOXC9 individually or in combination followed by retinoic acid (RA) mediated differentiation for 3 days. (Right panel) Bar graph shows the quantification of the average neurite branch length. Scale bar represents 50 μm. Data are presented as mean ± SD from three independent experiments. Two-way ANOVA with Tukey’s post hoc test was used, * p < 0.05, *** p < 0.001, **** p < 0.0001.
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    1) Product Images from "METTL3/MYCN cooperation drives m 6 A modification during trunk neural crest differentiation and represents a therapeutic vulnerability in MYCN-amplified neuroblastoma"

    Article Title: METTL3/MYCN cooperation drives m 6 A modification during trunk neural crest differentiation and represents a therapeutic vulnerability in MYCN-amplified neuroblastoma

    Journal: bioRxiv

    doi: 10.1101/2023.10.06.561194

    A, (Left panel) The total number of m 6 A peaks identified and (middle panel) identified motifs from de novo motif analysis of m 6 A peaks enriched in MYCN-amplified NB tumor samples. (Right panel) Metagene analysis showing relative m 6 A peak density at genes in both MYCN-amplified NB tumor samples. B, (Left panel) Immunoblot showing METTL3 expression in Dox induced (48 hours) control and METTL3 KD SK-N-BE and IMR-32 cells . GAPDH was used as a loading control. The values below indicate the fold change in levels of METTL3. (Right panel) Bar plots show cell viability of SK-N-BE and IMR-32 cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. C, (Left panel) Representative immunoblot showing simultaneous MYCN overexpression and METTL3 KD via Dox induction in SHEP (SHEP MYCN ) cells. GAPDH and vinculin were loading controls. The values below indicate the fold change in levels of METTL3 and MYCN. (Right panel) Bar plot shows the cell viability of SHEP MYCN cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. D, Representative immunoblot showing METTL3 and METTL14 expression following siRNA-mediated METTL3 KD. Vinculin was used as a loading control. The values below indicate the fold change in levels of METTL3 and METTL14. E, Representative images from colony formation assay performed in SK-N-BE cells with METTL3 KD (14 days post Dox induction). F, Top enriched terms associated with DEGs (shCtrl Vs. METTL3 KD) having m 6 A peaks (m 6 A+) in SK-N-BE cells. G, (Left panel) Brightfield images of SK-N-BE cells with stable overexpression of HOXC8 and HOXC9. (Right panel) Representative IF showing TUBB3 (green) and overexpression of MYC-tagged HOXC8 and HOXC9 (red) in SK-N-BE cells. H, (Left panel) Venn diagram comparison of HOXC9 target genes [genes with HOXC9 ChIP-seq peak and 1.5 fold change in expression between control vs HOXC9 overexpression] and differentially expressed between DEGs (shCtrl Vs. METTL3 KD) in SK-N-BE cells. (Right panel) Top enriched terms associated with the overlapping genes from the left panel. I, (Left panel) Interaction network of HOXC8 obtained using STRING-db with default parameters. (Right panel) Proximity ligation assay (PLA) showing the HOXC8 and HOXC9 PLA signal (green) between in SK-N-BE cell nucleus (marked by DAPI). The Negative control shows PLA with only the HOXC8 antibody. Scale bar represents 50 μm. J, (Left panel) Representative IF showing staining for PRPH and MYC-tagged HOXC8 and HOXC9 in SK-N-BE cell transiently overexpressing HOXC8 and HOXC9 individually or in combination followed by retinoic acid (RA) mediated differentiation for 3 days. (Right panel) Bar graph shows the quantification of the average neurite branch length. Scale bar represents 50 μm. Data are presented as mean ± SD from three independent experiments. Two-way ANOVA with Tukey’s post hoc test was used, * p < 0.05, *** p < 0.001, **** p < 0.0001.
    Figure Legend Snippet: A, (Left panel) The total number of m 6 A peaks identified and (middle panel) identified motifs from de novo motif analysis of m 6 A peaks enriched in MYCN-amplified NB tumor samples. (Right panel) Metagene analysis showing relative m 6 A peak density at genes in both MYCN-amplified NB tumor samples. B, (Left panel) Immunoblot showing METTL3 expression in Dox induced (48 hours) control and METTL3 KD SK-N-BE and IMR-32 cells . GAPDH was used as a loading control. The values below indicate the fold change in levels of METTL3. (Right panel) Bar plots show cell viability of SK-N-BE and IMR-32 cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. C, (Left panel) Representative immunoblot showing simultaneous MYCN overexpression and METTL3 KD via Dox induction in SHEP (SHEP MYCN ) cells. GAPDH and vinculin were loading controls. The values below indicate the fold change in levels of METTL3 and MYCN. (Right panel) Bar plot shows the cell viability of SHEP MYCN cells with METTL3 KD (6 days post Dox induction). Data are presented as mean ± SD from three independent experiments. Unpaired t -test was used, **** p < 0.0001. D, Representative immunoblot showing METTL3 and METTL14 expression following siRNA-mediated METTL3 KD. Vinculin was used as a loading control. The values below indicate the fold change in levels of METTL3 and METTL14. E, Representative images from colony formation assay performed in SK-N-BE cells with METTL3 KD (14 days post Dox induction). F, Top enriched terms associated with DEGs (shCtrl Vs. METTL3 KD) having m 6 A peaks (m 6 A+) in SK-N-BE cells. G, (Left panel) Brightfield images of SK-N-BE cells with stable overexpression of HOXC8 and HOXC9. (Right panel) Representative IF showing TUBB3 (green) and overexpression of MYC-tagged HOXC8 and HOXC9 (red) in SK-N-BE cells. H, (Left panel) Venn diagram comparison of HOXC9 target genes [genes with HOXC9 ChIP-seq peak and 1.5 fold change in expression between control vs HOXC9 overexpression] and differentially expressed between DEGs (shCtrl Vs. METTL3 KD) in SK-N-BE cells. (Right panel) Top enriched terms associated with the overlapping genes from the left panel. I, (Left panel) Interaction network of HOXC8 obtained using STRING-db with default parameters. (Right panel) Proximity ligation assay (PLA) showing the HOXC8 and HOXC9 PLA signal (green) between in SK-N-BE cell nucleus (marked by DAPI). The Negative control shows PLA with only the HOXC8 antibody. Scale bar represents 50 μm. J, (Left panel) Representative IF showing staining for PRPH and MYC-tagged HOXC8 and HOXC9 in SK-N-BE cell transiently overexpressing HOXC8 and HOXC9 individually or in combination followed by retinoic acid (RA) mediated differentiation for 3 days. (Right panel) Bar graph shows the quantification of the average neurite branch length. Scale bar represents 50 μm. Data are presented as mean ± SD from three independent experiments. Two-way ANOVA with Tukey’s post hoc test was used, * p < 0.05, *** p < 0.001, **** p < 0.0001.

    Techniques Used: Amplification, Western Blot, Expressing, Over Expression, Colony Assay, Comparison, ChIP-sequencing, Proximity Ligation Assay, Negative Control, Staining

    imr 32  (CLS Cell Lines Service GmbH)


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    Induction of p75NTR expression in <t>human</t> <t>neuroblastoma</t> cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
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    1) Product Images from "Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis"

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23073849

    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
    Figure Legend Snippet: Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

    Techniques Used: Expressing, Western Blot, Incubation

    The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.

    Techniques Used: Western Blot, Incubation, Expressing, Immunofluorescence, Microscopy, Staining, Activity Assay, Luminescence Assay, In Situ, TUNEL Assay

    Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.
    Figure Legend Snippet: Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.

    Techniques Used: Light Microscopy, Incubation, Western Blot

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    CLS Cell Lines Service GmbH imr-32 cells
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    CLS Cell Lines Service GmbH imr 32
    6p22.3 lncRNAs CASC15-003 and NBAT1 regulate MYCN levels in neuroblastoma (NB) cells. (A) Immunoblot shows the MYCN protein levels in vector control, CASC15-003 and NBAT1 overexpressing SK-N-BE(2), <t>IMR-32,</t> and SH-SY5Y cells (upper panel) and in Ctrl Sh, CASC15-003 and NBAT1 shRNA transduced IMR-32 and SH-SY5Y cells (lower panel). Vector control and Ctrl Sh were used as controls for overexpression and KD, respectively. GAPDH is used as a loading control. (B) Immunoblot of MYCN and USP36 in USP36 KD SK-N-BE(2), IMR-32, and SH-SY5Y cells. KD was carried out using lentiviral shRNA particles in SK-N-BE(2), IMR-32, and SH-SY5Y, whereas transient USP36 KD was performed using siRNA in SK-N-BE(2) (last panel on the right). Ctrl Sh or Ctrl Si was used as a control and GAPDH as a loading control. (C) Immunoblot of MYCN and USP36 in SK-N-BE(2), IMR-32, and SH-SY5Y, where USP36 was overexpressed from Halo-USP36 plasmid. Vector ctrl was used as a control and GAPDH as a loading control. (D) Immunoblot of MYCN protein levels in cycloheximide chase experiment. SK-N-BE(2) cells transfected with control or USP36 siRNA followed by treatment with cycloheximide (50 µg/mL) and harvested at the given time intervals. The values indicate the fold change in MYCN protein levels normalized to GAPDH. Ctrl Si was used as a control and GAPDH as a loading control for the immunoblot. (A–C) Immunoblots for MYCN in non-MNA SH-SY5Y cells were always exposed longer as compared to MNA cells for the detection of MYCN protein. The values indicate fold change in protein levels of MYCN or USP36 normalized to GAPDH.
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    1) Product Images from "NBAT1/CASC15-003/USP36 control MYCN expression and its downstream pathway genes in neuroblastoma"

    Article Title: NBAT1/CASC15-003/USP36 control MYCN expression and its downstream pathway genes in neuroblastoma

    Journal: Neuro-oncology Advances

    doi: 10.1093/noajnl/vdab056

    6p22.3 lncRNAs CASC15-003 and NBAT1 regulate MYCN levels in neuroblastoma (NB) cells. (A) Immunoblot shows the MYCN protein levels in vector control, CASC15-003 and NBAT1 overexpressing SK-N-BE(2), IMR-32, and SH-SY5Y cells (upper panel) and in Ctrl Sh, CASC15-003 and NBAT1 shRNA transduced IMR-32 and SH-SY5Y cells (lower panel). Vector control and Ctrl Sh were used as controls for overexpression and KD, respectively. GAPDH is used as a loading control. (B) Immunoblot of MYCN and USP36 in USP36 KD SK-N-BE(2), IMR-32, and SH-SY5Y cells. KD was carried out using lentiviral shRNA particles in SK-N-BE(2), IMR-32, and SH-SY5Y, whereas transient USP36 KD was performed using siRNA in SK-N-BE(2) (last panel on the right). Ctrl Sh or Ctrl Si was used as a control and GAPDH as a loading control. (C) Immunoblot of MYCN and USP36 in SK-N-BE(2), IMR-32, and SH-SY5Y, where USP36 was overexpressed from Halo-USP36 plasmid. Vector ctrl was used as a control and GAPDH as a loading control. (D) Immunoblot of MYCN protein levels in cycloheximide chase experiment. SK-N-BE(2) cells transfected with control or USP36 siRNA followed by treatment with cycloheximide (50 µg/mL) and harvested at the given time intervals. The values indicate the fold change in MYCN protein levels normalized to GAPDH. Ctrl Si was used as a control and GAPDH as a loading control for the immunoblot. (A–C) Immunoblots for MYCN in non-MNA SH-SY5Y cells were always exposed longer as compared to MNA cells for the detection of MYCN protein. The values indicate fold change in protein levels of MYCN or USP36 normalized to GAPDH.
    Figure Legend Snippet: 6p22.3 lncRNAs CASC15-003 and NBAT1 regulate MYCN levels in neuroblastoma (NB) cells. (A) Immunoblot shows the MYCN protein levels in vector control, CASC15-003 and NBAT1 overexpressing SK-N-BE(2), IMR-32, and SH-SY5Y cells (upper panel) and in Ctrl Sh, CASC15-003 and NBAT1 shRNA transduced IMR-32 and SH-SY5Y cells (lower panel). Vector control and Ctrl Sh were used as controls for overexpression and KD, respectively. GAPDH is used as a loading control. (B) Immunoblot of MYCN and USP36 in USP36 KD SK-N-BE(2), IMR-32, and SH-SY5Y cells. KD was carried out using lentiviral shRNA particles in SK-N-BE(2), IMR-32, and SH-SY5Y, whereas transient USP36 KD was performed using siRNA in SK-N-BE(2) (last panel on the right). Ctrl Sh or Ctrl Si was used as a control and GAPDH as a loading control. (C) Immunoblot of MYCN and USP36 in SK-N-BE(2), IMR-32, and SH-SY5Y, where USP36 was overexpressed from Halo-USP36 plasmid. Vector ctrl was used as a control and GAPDH as a loading control. (D) Immunoblot of MYCN protein levels in cycloheximide chase experiment. SK-N-BE(2) cells transfected with control or USP36 siRNA followed by treatment with cycloheximide (50 µg/mL) and harvested at the given time intervals. The values indicate the fold change in MYCN protein levels normalized to GAPDH. Ctrl Si was used as a control and GAPDH as a loading control for the immunoblot. (A–C) Immunoblots for MYCN in non-MNA SH-SY5Y cells were always exposed longer as compared to MNA cells for the detection of MYCN protein. The values indicate fold change in protein levels of MYCN or USP36 normalized to GAPDH.

    Techniques Used: Western Blot, Plasmid Preparation, shRNA, Over Expression, Transfection

    USP36 interacts with and regulates MYCN protein. (A) Upper panel: Immunostaining analysis in IMR-32 cells shows the endogenous MYCN in green and nucleolar marker nucleophosmin (NPM1) in red, stained with anti-MYCN and anti-NPM1 antibodies, respectively. DAPI was used as the nuclear stain. Lower panel: Immunostaining of USP36 (green) and NPM1 (red) stained with anti-USP36 and anti-NPM1 antibodies, respectively, in IMR-32 cells. DAPI was the nuclear stain. Scale bar, 5 µm. (B) SH-SY5Y cells transiently transfected with Halo-USP36 and HA-MYCN expressing plasmids followed by immunostaining analysis. Upper panel: Immunostaining of Halo-USP36 and HA-MYCN stained with anti-Halo and anti-HA antibodies, respectively. DAPI was the nuclear stain. Lower panel: Immunostaining for Halo-USP36 and NPM1 stained with anti-Halo or anti-NPM1 antibodies, respectively. DAPI was the nuclear stain. Scale bar, 5 µm. (C) Co-immunoprecipitation (Co-IP) showing the interaction between MYCN and USP36. Left and middle panels show the endogenous MYCN immunoprecipitation using anti-MYCN antibody in SK-N-BE(2) and IMR-32 cells, respectively. Anti-MYCN and anti-USP36 antibodies detected the presence of MYCN and USP36 in the anti-MYCN immunoprecipitation, IP:MYCN (lane3 from left). Left and middle lanes represent 5 or 10% input and negative control IgG, respectively. GAPDH immunoblot rules out the non-specific interactions in the Co-IP. Right panel: shows interaction between ectopically expressed MYCN and USP36 in HeLa cells. MYCN and Halo-USP36 were ectopically expressed in HeLa cells and the lysates were subjected to immunoprecipitation with anti-MYCN antibody in IP:MYCN. Anti-MYCN and anti-Halo antibodies detected the presence of MYCN and USP36 in both IP:MYCN and input. IgG is the negative control for IP. (D) Ubiquitination of MYCN in NB cells. Upper panel: Immunoblot shows the presence of MYCN in lysates (lane 3) immunoprecipitated with anti-FK2 antibody in SK-N-BE(2) cells treated overnight with MG132, IP:FK2. IgG used as a negative control and GAPDH immunoblot served as a control for nonspecific binding. Lower panel: Ectopically expressed Halo-USP36 deubiquitinates HA-MYCN in HeLa cells. HA-MYCN and Halo-USP36 were ectopically expressed in Hela cells. Two days after transfection, cells were treated with MG132 overnight and subjected to IP with anti-HA antibody. Immunoblot shows the ubiquitinated HA-MYCN in IP:HA-MYCN detected by FK2 antibody. IP:HA-MYCN also shows the levels of immunoprecipitated HA-MYCN. Anti-HA, Anti-Halo and Anti-GAPDH detect the levels of ectopic MYCN, USP36 and endogenous GAPDH, respectively. (E) Immunoblot for ubiquitinated MYCN in USP36 stable KD or USP36 overexpressing SK-N-BE(2) cells. Cells were treated with MG132 overnight and MYCN was immunoprecipitated and analyzed by Immunoblot to check the ubiquitination status of MYCN. Upper panel: Lysates from IMR-32 cells, stably transduced with Ctrl or USP36 Sh1 and Sh3, were immunoprecipitated with anti-MYCN antibody. Immunoblot shows the ubiquitination of immunoprecipitated MYCN in IP:MYCN detected by FK2 antibody. Also, IP:MYCN shows the levels of immunoprecipitated MYCN. Anti-MYCN, anti-USP36, and anti-GAPDH antibodies detect the MYCN, USP36, and GAPDH levels, respectively, in IP:MYCN and Input. IgG is the negative control for IP. Lower panel: MYCN was immunoprecipitated from HeLa cells, ectopically expressing vector Ctrl or Halo-USP36. Immunoblot shows the ubiquitinated MYCN detected by FK2 antibody and the corresponding levels of MYCN detected by anti-MYCN antibody in IP:MYCN. Anti-Halo, anti-MYCN and anti-GAPDH antibodies detect the levels of Halo-USP36, MYCN, and GAPDH, respectively, in the input.
    Figure Legend Snippet: USP36 interacts with and regulates MYCN protein. (A) Upper panel: Immunostaining analysis in IMR-32 cells shows the endogenous MYCN in green and nucleolar marker nucleophosmin (NPM1) in red, stained with anti-MYCN and anti-NPM1 antibodies, respectively. DAPI was used as the nuclear stain. Lower panel: Immunostaining of USP36 (green) and NPM1 (red) stained with anti-USP36 and anti-NPM1 antibodies, respectively, in IMR-32 cells. DAPI was the nuclear stain. Scale bar, 5 µm. (B) SH-SY5Y cells transiently transfected with Halo-USP36 and HA-MYCN expressing plasmids followed by immunostaining analysis. Upper panel: Immunostaining of Halo-USP36 and HA-MYCN stained with anti-Halo and anti-HA antibodies, respectively. DAPI was the nuclear stain. Lower panel: Immunostaining for Halo-USP36 and NPM1 stained with anti-Halo or anti-NPM1 antibodies, respectively. DAPI was the nuclear stain. Scale bar, 5 µm. (C) Co-immunoprecipitation (Co-IP) showing the interaction between MYCN and USP36. Left and middle panels show the endogenous MYCN immunoprecipitation using anti-MYCN antibody in SK-N-BE(2) and IMR-32 cells, respectively. Anti-MYCN and anti-USP36 antibodies detected the presence of MYCN and USP36 in the anti-MYCN immunoprecipitation, IP:MYCN (lane3 from left). Left and middle lanes represent 5 or 10% input and negative control IgG, respectively. GAPDH immunoblot rules out the non-specific interactions in the Co-IP. Right panel: shows interaction between ectopically expressed MYCN and USP36 in HeLa cells. MYCN and Halo-USP36 were ectopically expressed in HeLa cells and the lysates were subjected to immunoprecipitation with anti-MYCN antibody in IP:MYCN. Anti-MYCN and anti-Halo antibodies detected the presence of MYCN and USP36 in both IP:MYCN and input. IgG is the negative control for IP. (D) Ubiquitination of MYCN in NB cells. Upper panel: Immunoblot shows the presence of MYCN in lysates (lane 3) immunoprecipitated with anti-FK2 antibody in SK-N-BE(2) cells treated overnight with MG132, IP:FK2. IgG used as a negative control and GAPDH immunoblot served as a control for nonspecific binding. Lower panel: Ectopically expressed Halo-USP36 deubiquitinates HA-MYCN in HeLa cells. HA-MYCN and Halo-USP36 were ectopically expressed in Hela cells. Two days after transfection, cells were treated with MG132 overnight and subjected to IP with anti-HA antibody. Immunoblot shows the ubiquitinated HA-MYCN in IP:HA-MYCN detected by FK2 antibody. IP:HA-MYCN also shows the levels of immunoprecipitated HA-MYCN. Anti-HA, Anti-Halo and Anti-GAPDH detect the levels of ectopic MYCN, USP36 and endogenous GAPDH, respectively. (E) Immunoblot for ubiquitinated MYCN in USP36 stable KD or USP36 overexpressing SK-N-BE(2) cells. Cells were treated with MG132 overnight and MYCN was immunoprecipitated and analyzed by Immunoblot to check the ubiquitination status of MYCN. Upper panel: Lysates from IMR-32 cells, stably transduced with Ctrl or USP36 Sh1 and Sh3, were immunoprecipitated with anti-MYCN antibody. Immunoblot shows the ubiquitination of immunoprecipitated MYCN in IP:MYCN detected by FK2 antibody. Also, IP:MYCN shows the levels of immunoprecipitated MYCN. Anti-MYCN, anti-USP36, and anti-GAPDH antibodies detect the MYCN, USP36, and GAPDH levels, respectively, in IP:MYCN and Input. IgG is the negative control for IP. Lower panel: MYCN was immunoprecipitated from HeLa cells, ectopically expressing vector Ctrl or Halo-USP36. Immunoblot shows the ubiquitinated MYCN detected by FK2 antibody and the corresponding levels of MYCN detected by anti-MYCN antibody in IP:MYCN. Anti-Halo, anti-MYCN and anti-GAPDH antibodies detect the levels of Halo-USP36, MYCN, and GAPDH, respectively, in the input.

    Techniques Used: Immunostaining, Marker, Staining, Transfection, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Western Blot, Binding Assay, Stable Transfection, Transduction, Plasmid Preparation

    USP36 KD promotes tumor suppression. (A) MTT assay represents the proliferation status of IMR-32 and SK-N-BE(2) cells stably transduced with Ctrl or USP36 Sh1 and Sh3. Equal number of Ctrl or USP36Sh cells were seeded and allowed to proliferate for 48 h and then MTT assay was performed. (B) Representative images of the colony formation assay in IMR-32 and SK-N-BE(2) cells stably transduced with Ctrl or USP36 Sh1 and Sh3. Equal number of Ctrl or USP36Sh cells were seeded and Colony formation assay was performed 5 days after plating. (C,D) IMR-32 cells stably transduced with ctrl or USP36 ShRNA were subcutaneously injected to develop xenografts in mice. Tumors were harvested 40 days postinjection in mice. (C) Upper panel: Representative images of the tumors depicting the tumor size from xenografts ( n = 4). Lower panel: Box plots indicate the average weight (left) and volume (right) of the tumors from the upper panel. (D) Upper panel: representative images of the Ki67 staining on the xenografts derived from stable Ctrl or USP36 Sh3 IMR-32 cells. Short arrow heads indicate the representative Ki67-positive cells. Scale bar, 50 µm. Lower panel: Box plot shows the percentage of Ki67-positive cells counted from 3 different fields in Ctrl or USP36 Sh3 xenografts. For box plots (C and D), middle line represents the median and the box limits are 25th and 75th percentiles, whiskers are nearer quartile ± 1.5 times interquartile range. For A, C, and D, values represent mean +SD. * P < .05, ** P < .01, and *** P < .001 by Student’s t -test (2-tailed, 2-sample unequal variance).
    Figure Legend Snippet: USP36 KD promotes tumor suppression. (A) MTT assay represents the proliferation status of IMR-32 and SK-N-BE(2) cells stably transduced with Ctrl or USP36 Sh1 and Sh3. Equal number of Ctrl or USP36Sh cells were seeded and allowed to proliferate for 48 h and then MTT assay was performed. (B) Representative images of the colony formation assay in IMR-32 and SK-N-BE(2) cells stably transduced with Ctrl or USP36 Sh1 and Sh3. Equal number of Ctrl or USP36Sh cells were seeded and Colony formation assay was performed 5 days after plating. (C,D) IMR-32 cells stably transduced with ctrl or USP36 ShRNA were subcutaneously injected to develop xenografts in mice. Tumors were harvested 40 days postinjection in mice. (C) Upper panel: Representative images of the tumors depicting the tumor size from xenografts ( n = 4). Lower panel: Box plots indicate the average weight (left) and volume (right) of the tumors from the upper panel. (D) Upper panel: representative images of the Ki67 staining on the xenografts derived from stable Ctrl or USP36 Sh3 IMR-32 cells. Short arrow heads indicate the representative Ki67-positive cells. Scale bar, 50 µm. Lower panel: Box plot shows the percentage of Ki67-positive cells counted from 3 different fields in Ctrl or USP36 Sh3 xenografts. For box plots (C and D), middle line represents the median and the box limits are 25th and 75th percentiles, whiskers are nearer quartile ± 1.5 times interquartile range. For A, C, and D, values represent mean +SD. * P < .05, ** P < .01, and *** P < .001 by Student’s t -test (2-tailed, 2-sample unequal variance).

    Techniques Used: MTT Assay, Stable Transfection, Transduction, Colony Assay, shRNA, Injection, Staining, Derivative Assay

    imr 32  (CLS Cell Lines Service GmbH)


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    human neuroblastoma cells imr 32  (CLS Cell Lines Service GmbH)


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