Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Organisation of the Gnas cluster. First exons of protein coding transcripts are shown as filled boxes and first exons of noncoding transcripts as shaded boxes. Arrows show the initiation and direction of transcription. Nesp is transcribed across the cluster. The arrow corresponding to the paternal Gnas allele is shown as a dotted line to indicate that the paternal Gnas allele is repressed tissue specifically. The 10 kb and 12.6 kb lines represent the new truncation mutations Nespas-T int2 and Nespas-T int3 . Nespas non-coding transcripts are shown as grey lines. * represents the termination of Nespas transcription, approximately 30 kb from the Nespas promoter. Paternally expressed microRNAs are shown as vertical lines. Filled circles represent methylated regions. ICR represents the imprinting control region.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas before the Nesp promoter in Nespas intron 2/ Nesp exon 2. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette . The targeting vector shows the location of a 1.2 kb fragment (black box labelled Ap) from the rabbit β-globin gene to truncate Nespas and the position of the selection cassette (TKneopA URA3), flanked by loxP sites (open triangles). The targeted allele was designated Nespas-T int2 allele after Cre-mediated excision of the selectable marker cassette. Methylated DMRs are shown by filled circles. A, Afl II; X, Xho I; ICR, Imprinting Control Region. The arrow shows the approximate position of the polyadenylation cassette (pA) to truncate Nespas in exon 1 in Nespas-T ex1 ; ( b ) Southern analysis of ES cell DNA showing correct targeting in the Ap- neo allele. Ap- neo targeted clones were identified by the presence of a 12.0 kb Afl II fragment detected with the 5' external probe, probe A. Correct targeting at the 3' end was confirmed by the detection of a 14.2 kb Xho I fragment with Probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion; ( e ) Nespas is truncated and abundant in +/ Nespas-T int2 ; ( f ) Bisulphite analysis showing the Nesp DMR is unmethylated on the paternal allele in + SD2 / Nespas-T int2 . All clones from one individual are grouped into a block and two + SD2 / Nespas-T int2 individuals, (1) and (2), and one + SD2 /+ individual were analysed. Each row of circles represents a clone derived from the paternal allele and each circle corresponds to a separate CpG (filled circle, methylated CpG; unfilled circle, unmethylated CpG) for nucleotides 140,450–140,800; ( g ) The Nesp level is unaffected in +/ Nespas-T int2 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Plasmid Preparation, Selection, Marker, Methylation, Control, Clone Assay, Blocking Assay, Derivative Assay

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Transcription and methylation status of the Gnas cluster is unaltered on paternal inheritance of Nesp trun . ( a ) Schematic summary of the transcriptional and methylation status of Nesp and Nespas on the paternal allele in wild-type and +/ Nesp trun . The pA insertion will truncate Nesp if expressed. Row of filled circles, methylated allele; row of unfilled circles, unmethylated allele; ( b ) The level of Nespas exon 1 spliced onto exon 2 is unaltered in +/ Nesp trun ; ( c ) Methylation at the Nesp DMR is unaffected. PCR products from bisulphite-treated DNAs were separated on 3% agarose gels as either undigested (U) or digested with Taq 1 (T). Digestion products in the T lanes represent methylation of the CpG dinucleotide in the Taq 1 recognition sequence. Genomic DNA from +/ Nespas-T int2 in which both alleles were unmethylated was included as a negative control; ( d ) The levels of Nesp , Gnasxl and Exon1A ( Ex1A ) are unaffected.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Sequencing, Negative Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas after the Nesp promoter in Nespas intron 4. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette (black box labelled Ap) to truncate Nespas and the position of the selection cassette flanked by loxP sites (open triangles). The selection cassette was deleted on germline transmission and the targeted allele was designated Nespas-T int3 . A, Afl II; B, Bsr GI; X, Xho I; ( b ) Southern analysis of ES cell DNA showing correct targeting. Correctly targeted clones were identified by the presence of a 14.1 kb Afl II fragment detected with the 5′ external probe, probe A. Correct targeting at the 3′ end was confirmed by the detection of a 16.3 kb Bsr GI fragment with probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion. There was a greater fold difference in the spliced form (exon 1 spliced onto exon 2) than the unspliced form (intron 4); ( e ) Nespas is truncated and abundant in +/ Nespas-T int3 ; ( f ) Northern blot assay of Nespas and loading control Actb ; ( g ) Light Scanner melt profile, using a SNP in Nespas exon 3, to show Nespas is expressed from the paternal allele in +/ Nespas-T int3 ; ( h ) Bisulphite analysis showing the Nesp DMR is unmethylated on the maternal allele and methylated on the paternal allele in + SD2 / NespasT int3 ; ( i ) The Nesp level is unaffected in +/ Nespas-T int3 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Selection, Transmission Assay, Clone Assay, Northern Blot, Control, Methylation

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Levels of Gnasxl , Exon1A and Gnas in + / Nespas-T int2 and +/ Nespas-T int3 . ( a ) and ( b ) Gnasxl is downregulated in +/ Nespas-T int2 whereas Exon1A ( Ex1A ) and Gnas are unaffected; ( c ) and ( d ) Gnasxl , Exon1A and Gnas are unaffected in +/ Nespas-T int3 . Gnasxl and Ex1A levels were measured in newborn brain whereas Gnas and Ex1A were analysed in brown fat, a tissue in which Gnas is predominantly expressed from the maternal allele [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques:

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Summary of the transcriptional and methylation status of the paternal allele of the imprinted Gnas cluster in wild-type and mutant mice. ( a ) wild-type; ( b ) +/ Nespas-T ex1 ; ( c ) +/ Nespas-T int2 ; ( d ) +/ Nesp trun ; ( e ) +/ Nespas-T int3 . The Ap insertion truncates Nespas and the pA insertion will truncate Nesp if expressed. The approximate positions of the differentially methylated regions (DMRs) are shown by rows of filled circles on the methylated allele and unfilled circles on the unmethylated allele. Arrows show the start and direction of transcription, with thin arrows indicating weak transcription. Multiple arrows in ( c ) and ( e ) show increased levels of truncated Nespas . Exons 2–12 of Gnas are not shown. The figure is not drawn to scale. Information for ( b ), +/ Nespas-T ex1 , taken from [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Mutagenesis

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: Workflow chart of the study. First, 125 early-stage HCC patients and 125 healthy controls were recruited to evaluate the potential value of anti-GNAS autoantibody for early detection of HCC by ELISA. GNAS protein expressions in 61 paired HCC or adjacent normal tissues were tested by IHC, subsequently, the levels of GNAS protein in three HCC cell lines and the normal liver cell line were investigated by Western blot. GEO datasets were inquired to explore the mRNA expression level of GNAS, meanwhile, the mRNA levels in three HCC cell lines and the normal liver cell line were detected by real-time PCR. Additionally, ICGC Data Portal was also queried for exploring the mutation frequency of GNAS in HCC tissues.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: The scatter plot of autoantibody to GNAS in different groups (A), and ROC curves of autoantibody to GNAS when distinguishing early HCC and different subgroups of HCC from healthy control (B, C, D, E, F). HCC, hepatocellular carcinoma; AFP, alpha-feto-protein; ELISA, Enzyme-linked immunosorbent assay; HCC(I), HCC patients of TNM stage I; HCC(II), HCC patients of TNM stage II; HCC_AFP (+), HCC patients of AFP value greater than or equal to 20 ng/mL; HCC_AFP (−), HCC patients of AFP value less than 20 ng/mL; Se, sensitivity; Sp, specificity; AUC, area under the receiver operating characteristic curve; 95 % confidence interval of AUC in brackets.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: The levels and frequency of autoantibody to GNAS evaluated by ELISA.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: the expression of GNAS evaluated by IHC.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Expressing

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: the expression of GNAS in tissue and cell lines. (A) Immunohistochemical staining of GNAS in HCC tissue and adjacent normal tissue slides. Positive stain pattern of GNAS in representative HCC tissue of TNM stage I, weak stain pattern of GNAS in the representative adjacent normal tissue of TNM stage I (40× and 200 × magnifications) (B) the cropped gel displaying protein levels of GNAS tested by western blotting in cell lines. The lanes of upper lines from left to right showed the GNAS protein levels in cell lines of L02, SNU449, HepG2, and Hep3b, and the following lanes were the expression levels of reference protein GADPH. The corresponding uncropped full-length gel can be found in Additional file 1. (C) the relative expression of GNAS to GAPDH in protein level from a normal liver cell line (L02) and three HCC cell lines (SUN449, HepG2, and Hep3b). (D) the relative expression of GNAS to GAPDH in mRNA level from normal liver cell line (L02) and three HCC cell lines (SUN449, HepG2 and Hep3b), **, P < 0.01, ***, P < 0.001. (E) the expressions of GNAS at the mRNA level from three datasets of the GEO database, numbers in horizontal-axis parentheses represent the sample size.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Heliyon

Article Title: Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

doi: 10.1016/j.heliyon.2023.e22627

Figure Lengend Snippet: Frequency of GNAS Mutations in HCC across 5 cohorts.

Article Snippet: IHC assay was performed by using mouse monoclonal antibody against GNAS (Proteintech, China, 1:100 dilution) according to the manufacturer's recommendations to analyze GNAS protein expression level.

Techniques: Virus

Journal: Cells

Article Title: MAGED2 Is Required under Hypoxia for cAMP Signaling by Inhibiting MDM2-Dependent Endocytosis of G-Alpha-S

doi: 10.3390/cells11162546

Figure Lengend Snippet: Reagent and tools.

Article Snippet: G protein alpha S/GNAS cDNA ORF Clone, Human, C-HA tag , Sino Biological Inc. , HG12069-CY.

Techniques: Produced, Recombinant, cAMP Assay, Competitive ELISA, Mutagenesis, Software

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Organisation of the Gnas cluster. First exons of protein coding transcripts are shown as filled boxes and first exons of noncoding transcripts as shaded boxes. Arrows show the initiation and direction of transcription. Nesp is transcribed across the cluster. The arrow corresponding to the paternal Gnas allele is shown as a dotted line to indicate that the paternal Gnas allele is repressed tissue specifically. The 10 kb and 12.6 kb lines represent the new truncation mutations Nespas-T int2 and Nespas-T int3 . Nespas non-coding transcripts are shown as grey lines. * represents the termination of Nespas transcription, approximately 30 kb from the Nespas promoter. Paternally expressed microRNAs are shown as vertical lines. Filled circles represent methylated regions. ICR represents the imprinting control region.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas before the Nesp promoter in Nespas intron 2/ Nesp exon 2. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette . The targeting vector shows the location of a 1.2 kb fragment (black box labelled Ap) from the rabbit β-globin gene to truncate Nespas and the position of the selection cassette (TKneopA URA3), flanked by loxP sites (open triangles). The targeted allele was designated Nespas-T int2 allele after Cre-mediated excision of the selectable marker cassette. Methylated DMRs are shown by filled circles. A, Afl II; X, Xho I; ICR, Imprinting Control Region. The arrow shows the approximate position of the polyadenylation cassette (pA) to truncate Nespas in exon 1 in Nespas-T ex1 ; ( b ) Southern analysis of ES cell DNA showing correct targeting in the Ap- neo allele. Ap- neo targeted clones were identified by the presence of a 12.0 kb Afl II fragment detected with the 5' external probe, probe A. Correct targeting at the 3' end was confirmed by the detection of a 14.2 kb Xho I fragment with Probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion; ( e ) Nespas is truncated and abundant in +/ Nespas-T int2 ; ( f ) Bisulphite analysis showing the Nesp DMR is unmethylated on the paternal allele in + SD2 / Nespas-T int2 . All clones from one individual are grouped into a block and two + SD2 / Nespas-T int2 individuals, (1) and (2), and one + SD2 /+ individual were analysed. Each row of circles represents a clone derived from the paternal allele and each circle corresponds to a separate CpG (filled circle, methylated CpG; unfilled circle, unmethylated CpG) for nucleotides 140,450–140,800; ( g ) The Nesp level is unaffected in +/ Nespas-T int2 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Plasmid Preparation, Selection, Marker, Methylation, Control, Clone Assay, Blocking Assay, Derivative Assay

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Transcription and methylation status of the Gnas cluster is unaltered on paternal inheritance of Nesp trun . ( a ) Schematic summary of the transcriptional and methylation status of Nesp and Nespas on the paternal allele in wild-type and +/ Nesp trun . The pA insertion will truncate Nesp if expressed. Row of filled circles, methylated allele; row of unfilled circles, unmethylated allele; ( b ) The level of Nespas exon 1 spliced onto exon 2 is unaltered in +/ Nesp trun ; ( c ) Methylation at the Nesp DMR is unaffected. PCR products from bisulphite-treated DNAs were separated on 3% agarose gels as either undigested (U) or digested with Taq 1 (T). Digestion products in the T lanes represent methylation of the CpG dinucleotide in the Taq 1 recognition sequence. Genomic DNA from +/ Nespas-T int2 in which both alleles were unmethylated was included as a negative control; ( d ) The levels of Nesp , Gnasxl and Exon1A ( Ex1A ) are unaffected.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Sequencing, Negative Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas after the Nesp promoter in Nespas intron 4. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette (black box labelled Ap) to truncate Nespas and the position of the selection cassette flanked by loxP sites (open triangles). The selection cassette was deleted on germline transmission and the targeted allele was designated Nespas-T int3 . A, Afl II; B, Bsr GI; X, Xho I; ( b ) Southern analysis of ES cell DNA showing correct targeting. Correctly targeted clones were identified by the presence of a 14.1 kb Afl II fragment detected with the 5′ external probe, probe A. Correct targeting at the 3′ end was confirmed by the detection of a 16.3 kb Bsr GI fragment with probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion. There was a greater fold difference in the spliced form (exon 1 spliced onto exon 2) than the unspliced form (intron 4); ( e ) Nespas is truncated and abundant in +/ Nespas-T int3 ; ( f ) Northern blot assay of Nespas and loading control Actb ; ( g ) Light Scanner melt profile, using a SNP in Nespas exon 3, to show Nespas is expressed from the paternal allele in +/ Nespas-T int3 ; ( h ) Bisulphite analysis showing the Nesp DMR is unmethylated on the maternal allele and methylated on the paternal allele in + SD2 / NespasT int3 ; ( i ) The Nesp level is unaffected in +/ Nespas-T int3 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Selection, Transmission Assay, Clone Assay, Northern Blot, Control, Methylation

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Levels of Gnasxl , Exon1A and Gnas in + / Nespas-T int2 and +/ Nespas-T int3 . ( a ) and ( b ) Gnasxl is downregulated in +/ Nespas-T int2 whereas Exon1A ( Ex1A ) and Gnas are unaffected; ( c ) and ( d ) Gnasxl , Exon1A and Gnas are unaffected in +/ Nespas-T int3 . Gnasxl and Ex1A levels were measured in newborn brain whereas Gnas and Ex1A were analysed in brown fat, a tissue in which Gnas is predominantly expressed from the maternal allele [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques:

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Summary of the transcriptional and methylation status of the paternal allele of the imprinted Gnas cluster in wild-type and mutant mice. ( a ) wild-type; ( b ) +/ Nespas-T ex1 ; ( c ) +/ Nespas-T int2 ; ( d ) +/ Nesp trun ; ( e ) +/ Nespas-T int3 . The Ap insertion truncates Nespas and the pA insertion will truncate Nesp if expressed. The approximate positions of the differentially methylated regions (DMRs) are shown by rows of filled circles on the methylated allele and unfilled circles on the unmethylated allele. Arrows show the start and direction of transcription, with thin arrows indicating weak transcription. Multiple arrows in ( c ) and ( e ) show increased levels of truncated Nespas . Exons 2–12 of Gnas are not shown. The figure is not drawn to scale. Information for ( b ), +/ Nespas-T ex1 , taken from [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Mutagenesis