Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: IGF2 levels, Igf2 expression, and H19 ICR methylation in the cerebrum, liver, and plasma from 6- and 12-month-old male and female WT and 5xFAD mice. ( a ) Schematic representation of the Igf2/H19 gene cluster (see text for details). Parental-specific DNA methylation of the H19 ICR defines the imprinted status of both H19 and Igf2. Igf2 expression on the paternal allele (upper row) is associated with H19 ICR DNA methylation, H19 silencing, and an unmethylated Igf2 DMR2. Conversely, Igf2 silencing on the maternal allele (lower row) is associated with an unmethylated H19 ICR, transcription factor (CTCF) binding, H19 expression, and Igf2 DMR2 DNA methylation. ( b – d ) ELISA levels of IGF2, ( e – f ) RT-qPCR levels of Igf2 mRNA, and ( g – i ) GlucMS-qPCR levels of H19 ICR DNA methylation (5mC) in the cerebrum, liver, and plasma of female and male 5xFAD and WT mice at 6- and 12-months of age. Data are expressed as means + / − SEM. IGF2 /Igf2 , insulin-like growth factor 2; DMR2, differentially methylated region 2; ICR, imprinting control region; Me, DNA methylation; CTCF, CCCTC-binding factor; rpl13a , ribosomal protein L13a .; HpaII/Msp1, DNA restriction enzymes. See supplemental materials for results of statistical analyses.

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Expressing, Methylation, Clinical Proteomics, DNA Methylation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: Epigenetic marks associated with Aβ 42 binding to Igf2 DMR2 in mice. ( a ) Schematic representation of the mouse Igf2 promoter region (also see Fig. a). Mouse Igf2 has four promoters (P, 0–3) and three DMRs (0–2). Beneath is shown the Igf2 DMR2 DNA sequence we analyzed, with the location of seven CpG sites (bold) relative to the predicted Aβ 42 interacting domain (A β ID; boxed area). ( b ) ChIP-qPCR analyses of Aβ 42 association with H19 ICR and Igf2 DMR2 in cerebrum from 12-month-old 5xFAD mice. ChIP- and double ChIP-qPCR analyses of DNA methylation (5mC) and Aβ 42 association with Igf2 DMR2 in ( c ) cerebrum, ( d ) liver, and ( e ) plasma of male and female, 5xFAD and WT, 6- and 12-month-old mice. ChIP-qPCR analyses of H3K9Ac ( f – h ) and H3K9me3 ( i – k ) association with Igf2 DMR2 in cerebrum, liver, and plasma of male and female, 5xFAD and WT, 6- and 12-month-old mice. Data are expressed as means − / + SEM. Igf2 , insulin-like growth factor 2; DMR2, differentially methylated region 2; ICR, imprinting control region; 5mC, 5-methylcytosine; Aβ 42 , amyloid beta 1–42; H3K9Ac, histone 3 lysine-9 acetylation; H3K9me3, histone 3 lysine-9 tri-methylation.

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Binding Assay, Sequencing, ChIP-qPCR, DNA Methylation Assay, Clinical Proteomics, Methylation, Control

Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: Epigenetic marks associated with Aβ 42 binding to Igf2 DMR2 in human frontal cortex. ( a ) Schematic representation of the human Igf2 promoter region (also see Fig. a). Human Igf2 has five promoters (human P1, HuP1; and P, 0–3) and two DMRs (0 and 2). There is no mouse homologue for HuP1; however, P0–3, DMR0 and DMR2 are homologous in human and mouse. Beneath is shown the Igf2 DMR2 DNA sequence we analyzed, with the location of twelve CpG sites (bold) relative to the predicted Aβ 42 interacting domain (A β ID; boxed area). ( b ) ChIP-qPCR analyses of Aβ 42 association with H19 ICR and Igf2 DMR2 in AD and non-AD patients. ChIP- and double ChIP-qPCR analyses of DNA methylation (5mC) and Aβ 42 association with the Igf2 DMR2 in male and female, AD and non-AD patients. ChIP-qPCR analyses of H3K9Ac ( d ) and H3K9me3 ( e ) association with Igf2 DMR2 in male and female, AD and non-AD patients. Data are expressed as means + / − SEM.

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Binding Assay, Sequencing, ChIP-qPCR, DNA Methylation Assay

Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: IGF2 levels, Igf2 expression, H19 ICR methylation, and Aβ 40 and Aβ 42 levels in the frontal cortex of male and female AD and non-AD patients. ( a ) ELISA levels of IGF2, ( b ) RT-qPCR levels of Igf2 mRNA, ( c ) GlucMS-qPCR levels of H19 ICR DNA methylation (5mC), and ELISA levels of ( d ) Aβ 40 , ( e ) Aβ 42 , and ( f ) the Aβ 42 / Aβ 40 ratio in the frontal cortex of male and female, AD and non-AD patients. Data are expressed as means + / − SEM. IGF2 /Igf2 , insulin-like growth factor 2; rpl13a , ribosomal protein L13a; HpaII /Msp1, DNA restriction enzymes ; Aβ 40 , amyloid beta 1–40; Aβ 42 , amyloid beta 1–42.

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Expressing, Methylation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, DNA Methylation Assay

Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: Effects of Aβ 42 on Igf2 regulation and IGF2 levels in HEK239 cells. Cell cultures received a single treatment of Aβ 42 oligomer solution or saline vehicle, which were replaced every 3 days (removing Aβ 42 from the media), and the cells were harvested after 3-, 6-, or 9-days for in vitro assays. ( a ) ELISA levels of Aβ 42 from nuclear extracts in Aβ 42 -treated cultures. ( b ) ChIP-qPCR analyses of Aβ 42 association with H19 ICR and Igf2 DMR2 (containing the AβID ) 6-days following Aβ 42 -treatment. ChIP-qPCR analyses of ( c ) Aβ 42 , ( d ) DNA methylation (5mC), ( e ) H3K9Ac, and ( f ) H3K9me3 association with Igf2 DMR2 in Aβ 42 -treated cultures. ( g ) RT-qPCR analyses of Igf2 mRNA levels and ( h ) ELISA analysis of IGF2 levels in Aβ 42 -treated cultures. Data are expressed as means + / − SEM (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Saline, In Vitro, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, DNA Methylation Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

doi: 10.1038/s41598-023-29248-x

Figure Lengend Snippet: Dynamics of histone modification, CpG methylation, and transcription factor binding that control Igf2 expression associated with Aβ 42 accumulation in AD. ( a ) In Non-AD (WT mice) : enhanced Igf2 expression on the paternal allele (upper row) is associated with histone hypermethylation (H3K9me3), DNA hypermethylation (5mC), and reduced transcription factor (CTCF) binding to the H19 ICR, with concomitant histone hyperacetylation (H3K9Ac) and DNA hypomethylation of Igf2 DMR2. Conversely, attenuated Igf2 expression on the maternal allele (lower row) is associated with histone hyperacetylation (H3K9Ac), DNA hypomethylation, and increased transcription factor (CTCF) binding to the H19 ICR, with concomitant histone hypermethylation (H3K9me3), and DNA hypermethylation (5mC) of Igf2 DMR2. In AD ( 5xFAD mice ): as in WT mice, attenuated Igf2 expression on the paternal allele (upper row) is associated with histone hypermethylation (H3K9me3), and DNA hypermethylation (5mC); however , enhanced transcription factor ( Aβ 42 ) binding to Igf2 DMR2 is associated with histone hypoacetylation (H3K9Ac), histone hypermethylation (H3K9me3), DNA hypermethylation (5mC) and attenuation of Igf2 expression. As in WT mice, attenuated Igf2 expression on the maternal allele (lower row) is associated with histone hyperacetylation (H3K9Ac), DNA hypomethylation (5mC), and increased transcription factor (CTCF) binding to the H19 ICR; however, enhanced transcription factor ( Aβ 42 ) binding to Igf2 DMR2 is associated with histone hypoacetylation (H3K9Ac), histone hypermethylation (H3K9me3), DNA hypermethylation (5mC) and further attenuation of Igf2 expression. In this context, the regions of DNA that bind Aβ 42 (i.e., A β ID , Aβ interacting domains) have repressive chromatin marks and increased Aβ 42 binding association . ( b ) Temporal effects of Aβ 42 on Igf2 regulation. In cultured cells: Increased Aβ 42 levels are associated with increased binding of Aβ 42 to, and DNA hypermethylation of, the Igf2 differentially methylated region 2 (DMR2) (3-Days). Persistent Igf2 DMR2 Aβ 42 binding and DNA hypermethylation combined with histone modifications (deacetylation and methylation) are associated with a stable reduction of Igf2 expression and IGF2 levels (6-Days, 9-Days). IGF2 /Igf2 , insulin growth factor 2; DMR2, differentially methylated region 2; ICR, imprinting control region; CTCF, CCCTC-binding factor, Aβ 42 , amyloid beta 1–42.

Article Snippet: Immunoprecipitation was then performed using rabbit polyclonal IgG antibodies against 5mC (AnaSpec Inc., cat# BI-MECY-0500), Aβ 42 (MilliporeSigma Co., cat# AB5078P), CTCF (MilliporeSigma Co., cat# 07-729), H3K9Ac (Diagenode, cat# C15410004), H3K9me3 (Diagenode, cat# C15410193), as well as the control normal rabbit IgG (Santa Cruz Biotechnology Inc, cat# sc-2027).

Techniques: Modification, CpG Methylation Assay, Binding Assay, Control, Expressing, Cell Culture, Methylation

Journal: PLoS ONE

Article Title: Evidence of Specialized Tissue in Human Interatrial Septum: Histological, Immunohistochemical and Ultrastructural Findings

doi: 10.1371/journal.pone.0113343

Figure Lengend Snippet: A. Labeling for HCN4 (the heart obtained from a 58-year old male patient; brown color; a mouse monoclonal antibody, Alamone Labs, Israel; hematoxylin counterstain; ×50). Positive labeling of the specialized-like cells (†), and negative labeling of the typical contraction myocytes (*). A close up view shows sub-membrane immunolabeling with HCN4 of the T-like and P-like cells (a rabbit polyclonal antibody, Abcam, UK; hematoxylin counterstain; ×200). B. Labeling for Connexin43 (Cx43) (the heart obtained from a 58-year old male patient; brown color; hematoxylin counterstain; ×50). Notably, positive staining of the specialized-like cells, as well as of typical contraction myocytes, was found in 2 specimens. C. Labeling for Caveolin3 (brown color; hematoxylin counterstain; ×50). D. Double immunolabeling (primary antibody cocktail #1) for HCN4 (blue color, Abcam, UK) and Connexin43 (Cx43) (red color) showing co-localization of the proteins. The heart obtained from a 63-year old female patient; ×100. A co-localization coefficient HCN4:Cx43 = 1∶3. E. Double immunolabeling (primary antibody cocktail #2) for HCN4 (red color, Alamone Labs, Israel) and Caveolin3 (blue color) showing co-localization of the proteins (the heart obtained from a 63-year old female patient; ×400). A co-localization coefficient Caveolin3:HCN4 = 1∶4.

Article Snippet: Immunolabeling was performed with the following antibodies: a mouse monoclonal IgG against HCN4 at a dilution 1∶50 (clone S114-10, Abcam, UK), and a rabbit polyclonal antibody against HCN4 (Alamone Labs, Jerusalem, Israel); a rabbit polyclonal IgG against Connexin43 at a dilution 1∶50 (Diagnostic BioSystems, USA) and a mouse monoclonal IgG against Caveolin3 at a dilution 1∶50 (clone C-2; Santa Cruze Biotechnology, USA).

Techniques: Labeling, Membrane, Immunolabeling, Staining