Journal:

Article Title: Saccharomyces cerevisiae DNA Polymerase ? and Polymerase ? Interact Physically and Functionally, Suggesting a Role for Polymerase ? in Sister Chromatid Cohesion

doi: 10.1128/MCB.23.8.2733-2748.2003

Figure Lengend Snippet: (A) Pol2 coimmunoprecipitates with Trf4. Coexpression of FLAG-Pol2 and His-Trf4 and immunoprecipitation protocols are described in Materials and Methods. Western blots of crude extract (labeled “I” for input protein) from insect cells expressing various combinations of Trf proteins and FLAG-Pol2 were probed with antibody against Trf4 or anti-FLAG Pol2 as indicated. Lane 1, no recombinant protein; lane 2, His-Trf4; lane 3, His-Trf4 plus FLAG-Pol2. These extracts were incubated with anti-FLAG beads. After washing of the beads, proteins that bound from extracts (labeled “B” for bound protein) were eluted by boiling. The proteins were analyzed on Western blots probed with antibody against Trf4 or anti-FLAG Pol2 as indicated on the right. Lane 4, no recombinant protein; lane 5, His-Trf4; lane 6, His-Trf4 plus FLAG-Pol2. (B) Pol2 coimmunoprecipitates with Trf5. Coexpression of FLAG-Pol2 and Trf5-His is described in Materials and Methods. Western blots of crude extract from insect cells are represented in the same order as those in Fig. ​Fig.2A.2A. (C) Recombinant Trf4 prepared in E. coli stimulates Pol ɛ holoenzyme. Trf4 was purified exactly as described previously (70). The oligo(dT)12-18 primer extension assay is described in Materials and Methods. Reaction mixtures contained 680 ng of Trf4, the amount required to observe Trf4 DNA polymerase activity (lanes 2 and 3), and/or 0.15 U of Pol ɛ, as indicated, are shown. The high level of Trf4 is saturating for stimulatory activity (see panel D). Lane 1, no protein; lane 2, Trf4-His with 0.1 mM dTTP; lane 3, Trf4-His with 1 mM dTTP; lane 4, Pol ɛ with 0.1 mM dTTP; lane 5, Pol ɛ with 1 mM dTTP; lane 6, Pol ɛ plus Trf4-His with 0.1 mM dTTP; and lane 7, Pol ɛ plus Trf4-His with 1 mM dTTP. (D) Titration of stimulatory activity of scTrf4 made in bacteria. The indicated amounts of scTrf4 were assayed for stimulation of [3H]dTMP incorporation by 0.15 U of Pol ɛ on an oligo(dT)-poly(dA) substrate as described in Materials and Methods. (E) scTrf4-His expressed in insect cell cochromatographs with Pol ɛ-stimulatory activity. scTrf4-His was expressed in insect cells. Silver staining of Trf4-His after purification through Ni2+-nitrilotriacetic acid and Mono Q columns, as described in Materials and Methods, and gel electrophoresis is shown at the top. Numbers refer to MonoQ fraction numbers. The same fractions are assayed for stimulation of primer extension by pol ɛ. Each fraction from the Mono Q column was dialyzed, and 2 μl of each fraction was used in a 20-μl reaction. Fraction 13 contained 17 ng of Trf4 protein (13 nM); but 2.7 nM Trf4 gave equivalent stimulation (not shown). The first lane shows no primer extension, the second lane shows activity of 0.15 U of Pol ɛ (0.5 nM) alone, and the subsequent lanes are the Mono Q fractions of the Trf4 purification assayed with 0.15 U of Pol ɛ (0.5 nm). The fraction numbers are identified above. (F) scTrf4-His and Pol ɛ-stimulatory activity copurify. [3H]dTMP incorporation assay: the same fractions from the Mono Q column were assayed for [3H]dTMP incorporation on an oligo(dT)-poly(dA) substrate as described in Materials and Methods in the presence of 0.15 U of Pol ɛ (0.5 nm). (G) Trf4 from the MonoQ column is highly purified. Coomassie-stained gel of Trf4 from fraction 14 of the MonoQ column. (H) scTrf4-His does not efficiently stimulate Pol2-140 lacking the C-terminal 1,000 amino acids. Three levels (0.075, 0.15, or 0.3 U) of either Pol ɛ (solid dots) or truncated Pol2 protein (open dots) were assayed with saturating amounts (40 ng) of Trf4-His purified from insect cells. Similar results were obtained with scTrf4 prepared in E. coli.

Article Snippet: As controls that the stimulation was not due to contaminating E. coli DNA polymerase I (the most abundant DNA polymerase in the bacterial extracts) in the scTrf4 preparation, we showed that DNA polymerase I of E. coli (Gibco BRL) did not stimulate Pol ɛ and antibody to E. coli Pol I (gift of S. Linn, U.C.

Techniques: Immunoprecipitation, Western Blot, Labeling, Expressing, Recombinant, Incubation, Purification, Primer Extension Assay, Activity Assay, Titration, Silver Staining, Nucleic Acid Electrophoresis, Staining

Journal: eLife

Article Title: MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface

doi: 10.7554/eLife.73348

Figure Lengend Snippet:

Article Snippet: recombinant DNA reagent , Mouse Ncaph2 cDNA , Origene , MC200537 , Initial cDNA used for cloning in pRNA.

Techniques: Plasmid Preparation, Isolation, Transfection, Construct, Produced, Recombinant, Clone Assay, In Vitro, Injection, CRISPR, Sequencing, Imaging, Labeling, Protein Purification, Silver Staining, Staining, Protease Inhibitor, In Vivo, Fluorescence, Amplification, Software

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2 and TET3 associate with the O -GlcNAc transferase OGT and promote GlcNAcylation. ( A ) Silver stain gel of HaloTag-TET protein complex isolations and HaloTag alone control (Ctrl). Protein pulldowns were performed from HEK293T cells overexpressing the indicated HT constructs (see Materials and methods and for details). As not all of the indicated complex isolations were performed at the same time, two separate silver stain gels were run, as shown in this panel. ( B ) Table of transcriptional or chromatin protein interactors found in the various HaloTag-TET isolations. Spectral counts for each interacting protein are shown for biological replicates. TET1, but not TET2, as previously reported ( ; ), shows interaction with SIN3A. OGT interacts with all TET proteins, though it is most highly abundant with TET2 and TET3. ( C ) Detection of OGT by western blotting from HT-TET2 and HT-TET3 pulldowns from ( A ). The indicated pulldowns were probed with an anti-OGT antibody to detect the presence of OGT. OGT and beta-Actin shown as input loading controls. ( D ) TET2 and TET3 co-immunoprecipitate (CoIP) with endogenous OGT from untransfected HEK293T cells. Cell extracts were immunoprecipitated with anti-OGT or rabbit IgG and probed with antibodies against the indicated proteins. An IP control of OGT alone is shown to demonstrate specific capture and enrichment of OGT. Inputs loading controls are shown for all. Note that in this experiment very weak expression of TET2 relative to TET3 is observed. ( E ) The global level of hmC does not change after cell treatment with Alloxan or PUGNAc. Dot blot quantification of global hmC after the indicated treatments. The hmC content is normalized with respect to the input DNA and to mock-treated cells, where the ratio is set at 1.00. Error bars indicate s.d. of three independent experiments. As controls, western blots using anti- O -GlcNAc antibody show the expected decrease in GlcNAcylation with Alloxan and increase with PUGNAc. HDAC1 input loading controls are also shown. Vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( F ) Global decrease in GlcNAcylation is observed in TET2/3 knockdowns. Left: TET2 kd or TET3 kd show decreased GlcNAc activity. Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl, RNAi TET2, or RNAi TET3, and UDP-[ 3 H]GlcNAc incorporation was measured. The amount incorporated into the control cells was set at 1. Error bars indicate s.d. of three independent experiments (* P <0.05). Right: Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl or RNAi TET2/3 and global GlcNAcylation was visualized with an antibody against O -GlcNAc. HDAC1 input loading control is also shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Silver Staining, Control, Construct, Western Blot, Immunoprecipitation, Expressing, Dot Blot, Activity Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3–OGT show genomic co-localization around TSSs and impact on H3K4me3 and transcriptional activation. ( A ) Left: Venn diagrams indicating significant overlap of TET2 and OGT bound regions (left part; P -value<10 −10 ) identified after HaloCHIP-Seq in HEK293T cells expressing HT-TET2, or HT-OGT. Right: TET2–OGT targets are primarily found at TSSs and CpG-rich sequences. Similar profiles were also observed for TET3–OGT . ( B ) An analysed subset of TET2–TET3–OGT targets show a lack of DNA methylation and hydroxymethylation, yet display GlcNAcylation. qPCR analysis of TET2–TET3–OGT binding and non-binding regions after MeDIP (top), hMeDIP (middle), or ChIP with an anti- O -GlcNAc antibody (bottom). ‘% Input' represents real-time qPCR values normalized with respect to the input chromatin. Known methylated and hydroxymethylated regions are shown as positive controls in MeDIP and hMeDIP panels.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Binding Assay, Methylated DNA Immunoprecipitation, Methylation

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: ( C ) TET2/3–OGT targets in HEK293T cells are enriched for H3K4me3 as depicted in a Venn diagram; P -value<10 −10 . ( D ) Examples of HaloCHIP-Seq OGT, TET2, TET3, and ChIP-Seq H3K4me3 profiles (UCSC tracks). ( E ) Decreased levels of H3K4me3 in TET2 kd cells. Upper-left: decrease in the normalized number of H3K4me3 reads in TET2/3–OGT-binding regions in TET2 kd cells versus control RNAi-treated cells. Upper-right: pie chart showing the percentage of TET2–TET3–OGT binding regions with a statistically significant reduction of the normalized number of H3K4me3 reads for TET2 kd versus control RNAi-treated cells. Lower-part: examples of H3K4me3 ChIP-Seq profiles (UCSC tracks) in TET2–TET3–OGT-binding regions for the RNAi control versus TET2 kd sample. ( F ) Western blot showing global decrease in H3K4me3 in a TET2/3 double kd cells. Lysates from mock HEK293T RNAi kd or TET2/3 kd cells were probed for H3K4me3 using an anti-H3K4 antibody in western blot. Tubulin is shown as a loading control. ( G ) OGT activity is important for H3K4me3. Cell extracts were prepared from HEK293T cells treated with or without the OGT inhibitor Alloxan, and then western blots for H3K4me3 were performed. HDAC1 and H3 are shown as loading controls and a western blot against O -GlcNAc was used to monitor specific GlcNAcylation inhibition by Alloxan. Vertical lines indicate juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( H ) Decreases in transcription are observed in both TET2/3 knockdowns and an OGT knockdown. The indicated target genes (which showed decrease in H3K4me3 after TET2 kd; cf. E ) and negative controls (unbound TET2/3–OGT–H3K4me3 targets), were analysed by RT–qPCR in HEK293T cells subjected to the various listed RNAi treatments. Independent experiments were performed in duplicates.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Control, Western Blot, Activity Assay, Inhibition, Knockdown, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin. ( A ) Mass spectrometry reveals HCF1, a known target of OGT and component of SET1/COMPASS , as an interacting partner of HT-TET2 and HT-TET3. Biological duplicates and respective spectral counts (SpC) for HCF1 are shown. ( B ) Protein pulldowns of HT-OGT coupled with mass spectrometry identify HCF1, TET2, TET3, and all components of SET1/COMPASS as partners of OGT. Biological duplicates and SpC for each protein identified are shown for HT-OGT and Ctrl isolations. ( C ) The interaction of HCF1 and SET1/COMPASS components with HT-OGT depends on O -GlcNAc activity. Plot showing average SpCs for HCF1 and SET1/COMPASS components isolated from HT-OGT pulldowns of untreated (grey bars) and Alloxan-treated (green bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( D ) The interaction of HT-SETD1A with SET1/COMPASS components and OGT is reduced by a TET2/3 double kd. Plot showing average SpCs for SET1/COMPASS components and OGT isolated from HT-SETD1A pulldowns of control RNAi-treated (grey bars) and TET2/3 kd (blue bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( E ) A significant reduction in HCF1 GlcNAcylation is observed after TET2/3 double kd. Upper diagram shows a schematic representation of full-length HCF1 and its domains . The GlcNAcylated peptides identified by mass spectrometry from HT-SETD1A isolations from control RNAi-treated and TET2/3 kd cells are indicated below. The full-length HCF1 amino-acid sequence (NP_005325.2) shows the corresponding GlcNAcylated peptides highlighted in yellow with RNAi Ctrl on the left and RNAi TET2/3 kd on the right. ( F ) Bioluminescence resonance energy transfer (BRET) assays show reduction of SETD1A binding to histone H3.3 in the presence of an OGT inhibitor and in TET2/3 kd cells. Upper diagram showing the schematic of BRET energy transfer upon binding of a NanoLuc-SETD1A fusion donor and fluorescently labelled Histone H3.3-HaloTag fusion acceptor in live HEK293T cells (see Materials and methods for experimental details and calculation of BRET). Left: BRET measurements were calculated without treatment (grey) or with Alloxan treatment (green). Right: BRET measurement for RNAi control (grey) or RNAi TET2/3 (blue). Biological triplicates ±s.d. are shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Mass Spectrometry, Isolation, Control, Sequencing, Bioluminescence Resonance Energy Transfer

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters. ( A ) Genome-wide co-localization of endogenous Tet2 with O -GlcNAc and H3K4me3 at promoters and CpG-rich regions. Venn diagrams are shown ( P -value overlap<10 −10 ) as well as the indicated genome-wide distribution. ( B ) Tet2, O -GlcNAc, and H3K4me3 are enriched at many active genes, mirroring the presence of RNA Pol II. Upper panel: Venn diagram showing the overlap of Tet2 and O -GlcNAc with RNA Pol II ( P -value overlap <10 −10 ); lower panel: Box plots showing the reads density at targets and non-targets (others) for ChIP-Seq RNA Pol II or RNA-Seq in mouse bone marrow. ( C ) Global decrease in GlcNAcylation is observed in Tet2 knockout mouse bone marrow. Mouse bone marrow tissues with or without a Tet2 knockout were analysed by western blot for O -GlcNAc levels using an anti- O -GlcNAc antibody. HDAC1 is shown as loading control. ( D ) ChIP-Seq for H3K4me3 in Tet2 knockout mouse tissues shows reduced global H3K4me3 at target promoters. Overall impact on H3K4me3 peak significance (−log( P -value) of the peaks) between wild-type and Tet2 knockout bone marrow is shown. ( E ) Table showing key haematopoietic genes with specifically reduced H3K4me3 in Tet2 knockout as compared to wild type. The location at CpG islands and the promoter class for each is listed. Lower part: example of H3K4me3 ChIP-Seq profiles (UCSC tracks) in wild type versus Tet2 knockout.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Knock-Out, Genome Wide, ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Model connecting DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. Based on our findings, a hierarchical model of the involved proteins, with the cascade of their respective activities, can be envisaged as followed: (1) The first sequence of events in the cascade is the formation of TET2/3–OGT interaction, which promotes OGT GlcNAcylation on numerous proteins, including HCF1; (2) In a TET-dependent manner, a GlcNAcylated HCF1 is important for the formation of the SET1/COMPASS; (3) In the last step, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Sequencing, Activity Assay, Binding Assay, Activation Assay

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( A ) Plasma membrane fractions were isolated from WT and IP6K1 KO MEFs. Silver stain revealed an enriched protein, Na + /K + -ATPase-α1, at ~100 kDa in IP6K1 KO preparations (arrow). ( B and C ) Na + /K + -ATPase-α1 protein level was increased in whole-cell lysates of IP6K1 KO MEFs ( n = 4; normalized to β-actin). ( D ) Na + /K + -ATPase-α1 and Na + /K + -ATPase-β1 protein levels in plasma membrane fractions of kidneys were increased in IP6K1 KOs. ( E ) Overexpression of myc-tagged IP6K1 decreased Na + /K + -ATPase-α1 and Na + /K + -ATPase-β1 protein levels in hRPTCs. ( F ) Immunostaining of Na + /K + -ATPase-α1 in WT and IP6K1 KO kidneys. Fluorescein-labeled Lotus tetragonolobus Lectin (LTL) stained brush border of proximal tubule cells, and Hoechst 33342 labeled nucleus. Na + /K + -ATPase-α1 is selectively increased in the renal tubule cells (arrows) but not in the glomerulus (arrowheads) of IP6K1 KOs. ( G ) Deletion of IP6K1 increased Na + /K + -ATPase activity ( n = 5; normalized to WT mean). ( H and I ) WT and IP6K1 KO mice were fed with high-salt (HS) diet (H) or normal-salt (NS) diet (I) for 9 weeks. (H) Daily urinary Na + excretion of HS diet–fed IP6K1 KO mice was less than that of HS diet–fed WT ( n = 6). (I) Daily urinary Na + excretions in NS diet–fed mice were similar ( n = 4). ( J and K ) Age-matched IP6K1 KO (J) and WT (K) mice were fed with HS or NS diet for 9 weeks. (J) HS diet–fed IP6K1 KO mice gained weight ( n = 7). (K) HS diet–fed WT mice did not gain weight ( n = 7).

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Clinical Proteomics, Membrane, Isolation, Silver Staining, Over Expression, Immunostaining, Labeling, Staining, Activity Assay

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( A ) WT and IP6K1 KO MEF cells were treated with cycloheximide (100 μM) for 16 hours to stop protein synthesis. Dimethyl sulfoxide (DMSO) was used as a solvent control. Deletion of IP6K1 slowed down Na + /K + -ATPase-α1 degradation. ( B ) Na + /K + -ATPase-α1 in WT and IP6K1 KO mice kidneys was immunoprecipitated (IP) and blotted for ubiquitin. Deletion of IP6K1 reduced Na + /K + -ATPase-α1 ubiquitination. ( C ) Cell membrane proteins of WT and IP6K1 KO MEF cells were labeled with biotin. The cells were incubated at 37°C for 4 hours then fixed with 4% paraformaldehyde. Na + /K + -ATPase-α1 was immunostained, and biotin was labeled with fluorescent streptavidin. In WT cells, colocalization of Na + /K + -ATPase-α1 with biotin was observed in the cytoplasm (inset), indicating normal endocytosis. In IP6K1 KO cells, biotin was observed in the cytoplasm (inset), but Na + /K + -ATPase-α1 remained in the cell membrane, suggesting that endocytosis of Na + /K + -ATPase-α1 was disrupted. Scale bars, 10 μm. ( D and E ) Coimmunoprecipitation of Na + /K + -ATPase-α1 and AP2β from WT and IP6K1 KO kidneys. (D) Less AP2β was co–pulled down by Na + /K + -ATPase-α1 in IP6K1 KOs. (E) Less Na + /K + -ATPase-α1 was coprecipitated by AP2β in IP6K1 KOs. ( F ) Endocytic vesicles of WT and IP6K1 KO kidneys were isolated and blotted for Na + /K + -ATPase-α1. The IP6K1 KO endocytic vesicles contained less amount of Na + /K + -ATPase-α1, suggesting that endocytosis of Na + /K + -ATPase-α1 was disrupted. ( G ) Quantification of Na + /K + -ATPase-α1 protein levels in endocytic vesicles of WT and IP6K1 KO kidneys ( n = 4; normalized to AP2β).

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Solvent, Control, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Labeling, Incubation, Isolation

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( A and B ) WT IP6K1, kinase-defective mutant (mut) IP6K1, or GFP (negative control) were overexpressed in IP6K1 KO MEF cells to rescue the expression of IP6K1. Overexpression of WT, but not mut IP6K1, decreased Na + /K + -ATPase-α1 protein level ( n = 3; normalized to β-actin). ( C ) Human embryonic kidney (HEK) 293 cells were treated with DMSO or N 2 -( m -(trifluoromethyl)benzyl) N 6 -( p -nitrobenzyl)purine (TNP; 3 μM) for 24 hours to inhibit 5-InsP 7 synthesis. TNP treatment increased Na + /K + -ATPase-α1 protein level. ( D ) HEK 293 cells were treated with increasing concentrations of TNP (0 μM, DMSO) for 24 hours. Na + /K + -ATPase-α1 was immunoprecipitated and blotted for ubiquitin. TNP treatment dose-dependently depleted Na + /K + -ATPase-α1 ubiquitination. ( E ) Flag-tagged Na + /K + -ATPase-α1 was overexpressed in HEK 293 cells. Cells were treated with DMSO or TNP (3 μM) for 24 hours. Flag-tagged Na + /K + -ATPase-α1 was immunoprecipitated by an anti-flag antibody and blotted for AP2β. The amount of coimmunoprecipitated AP2β was less in TNP-treated cells. ( F ) Glutathione S -transferase (GST)–fused AP2β (GST-AP2β) was overexpressed in HEK 293 cells. Cells were treated with DMSO or TNP (3 μM) for 24 hours. GST-AP2β was pulled down by Glutathione Sepharose and blotted for Na + /K + -ATPase-α1. Less Na + /K + -ATPase-α1 was pulled down in TNP-treated cells. ( G ) GFP-fused Na + /K + -ATPase-α1 was overexpressed in HEK 293 cells. Cells were incubated with DMSO or photocaged 5-InsP 7 for 4 hours and MitoTracker (red) for 30 min. Photochemical release of 5-InsP 7 by ultraviolet (UV) irradiation (dotted box) decreased GFP-Na + /K + -ATPase-α1 level in the plasma membrane. Nonilluminated neighboring cells were used as controls. Scale bar, 5 μm.

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Negative Control, Expressing, Over Expression, Immunoprecipitation, Ubiquitin Proteomics, Incubation, Irradiation, Clinical Proteomics, Membrane

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( A and B ) Coimmunoprecipitation of Na + /K + -ATPase-α1 and PI3K p85α from WT and IP6K1 KO mice kidneys. (A) Less PI3K p85α was co–pulled down by Na + /K + -ATPase-α1 in IP6K1 KO preparations. (B) Less Na + /K + -ATPase-α1 was coprecipitated by PI3K p85α in IP6K1 KO samples. ( C ) Flag-tagged Na + /K + -ATPase-α1 was overexpressed in HEK 293 cells. The cells were treated with TNP (3 μM) to block 5-InsP 7 synthesis for 24 hours. DMSO was used as the solvent control. Flag-tagged Na + /K + -ATPase-α1 was immunoprecipitated by an anti-flag antibody and blotted for PI3K p85α. The amount of coprecipitated PI3K p85α was less in TNP-treated cells. ( D ) GST-fused PI3K p85α (GST-p85α) was overexpressed in HEK 293 cells. The cells were treated with TNP (3 μM) for 24 hours. DMSO was used as the solvent control. GST-p85α was pulled down by Glutathione Sepharose and blotted for Na + /K + -ATPase-α1. Less Na + /K + -ATPase-α1 was co–pulled down in TNP-treated cells. ( E ) Myc-tagged IP6K1 was overexpressed in HEK 293 cells. Myc-tagged GFP was overexpressed in separate HEK 293 cells as negative control. Myc-tagged IP6K1 and myc-tagged GFP were pulled down by an anti-myc antibody. Myc-tagged IP6K1 co–pulled down PI3K p85α but not Na + /K + -ATPase-α1, AP2β, nor AP2μ. ( F and G ) Coimmunoprecipitation of IP6K1 and PI3K p85α in WT and IP6K1 KO kidneys. (F) PI3K p85α was coimmunoprecipitated by IP6K1 in WT preparations only. (G) IP6K1 was co–pulled down by PI3K p85α in WT samples.

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Blocking Assay, Solvent, Control, Immunoprecipitation, Negative Control

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( A and B ) Flag-tagged Na + /K + -ATPase-α1 immunoprecipitated on protein A/G beads was incubated with PI3K p85α in the presence of (A) increasing concentrations of 5-InsP 7 and (B) 5 μM 3-PCP, 5-PCP, or CF2. Binding between Na + /K + -ATPase-α1 and PI3K p85 was enhanced by 5-InsP 7 , 3-PCP, 5-PCP, and CF2. ( C ) PI3K p85α was incubated with control resin, 2-position–linked 5-PCP resin [5-PCP resin (2)], or 1/3-position–linked 5-PCP resin [5-PCP resin (1/3)]. PI3K p85α was pulled down by 5-PCP resin (1/3) but not by 5-PCP resin (2). ( D ) Flag-tagged domains of PI3K p85α (SH3, RhoGAP, nSH2, and cSH2) were overexpressed in HEK 293 cells. Whole-cell lysates were incubated with 5-PCP resin (1/3), which only pulled down flag-tagged RhoGAP. ( E ) Flag-tagged full-length (FL) PI3K p85α and flag-tagged RhoGAP domain–deleted (ΔRhoGAP) PI3K p85α were overexpressed in HEK 293 cells. Whole-cell lysates were incubated with 5-PCP resin (1/3), which pulled down flag-tagged FL PI3K p85α but not flag-tagged ΔRhoGAP PI3K p85α. ( F ) Flag-tagged FL PI3K p85α and flag-tagged ΔRhoGAP PI3K p85α immunoprecipitated on protein A/G beads were incubated with Na + /K + -ATPase-α1. Flag-tagged ΔRhoGAP PI3K p85α pulled down more Na + /K + -ATPase-α1 than flag-tagged FL PI3K p85α did (without CF2) and to a level similar to flag-tagged FL PI3K p85α in the presence of CF2. ( G to I ) Overexpression of flag-tagged ΔRhoGAP PI3K p85α in HEK 293 cells reduced Na + /K + -ATPase-α1 protein level in whole-cell lysates (WCLs) [(G) and (H), n = 3; normalized to β-actin] and membrane fractions (I).

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Incubation, Binding Assay, Control, Over Expression, Membrane

Journal: Science Advances

Article Title: The inositol pyrophosphate 5-InsP 7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α

doi: 10.1126/sciadv.abb8542

Figure Lengend Snippet: ( Top ) IP6K1 physiologically associates with PI3K p85α and generates a local pool of 5-InsP 7 , which binds the RhoGAP domain of PI3K p85α. Binding of 5-InsP 7 with PI3K p85α disinhibits the interaction between PI3K p85α and Na + /K + -ATPase-α1. This recruits AP2, which mediates clathrin-mediated endocytosis and leads to downstream degradation of Na + /K + -ATPase-α1. ( Bottom ) Depleting 5-InsP 7 disrupts the binding between PI3K p85α and Na + /K + -ATPase-α1, which impairs recruitment of endocytic machinery and downstream degradation.

Article Snippet: Antibodies against Na + /K + -ATPase-α1 and ubiquitin were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: The collagenases are able to cleave the PAR2 extracellular domain. A 42-amino-acid peptide corresponding to Arg31–Lys72 of the extracellular domain of PAR2 (denoted in red) was produced. Various known cleavage sites are highlighted: the canonical activation site (trypsin, matriptase, etc., with the tethered ligand/activator peptide sequence underlined); CS, cathepsin S; PR3, proteinase 3; CG, cathepsin G; NE, neutrophil elastase (A). The PAR231–72 peptide (10 μm) was incubated with 10 nm hepsin or elastase, 1 nm cathepsin G, or 0.1 nm matriptase for the indicated durations before resolving on 20% polyacrylamide gels utilizing a Tris-Tricine buffer system and silver staining. S, substrate; P, product. Presented gels are representative of at least two independent experiments (B). The PAR231–72 peptide (10 μm) was incubated with increasing concentrations of APMA-activated recombinant pro-MMP-1, -8, and -13 for 24 h before resolving on 20% polyacrylamide gels utilizing a Tris-Tricine buffer system and silver staining. S, substrate; P, product. The presented gels are representative of three independent experiments (C).

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Produced, Activation Assay, Sequencing, Incubation, Silver Staining, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: The MMP collagenases cleave PAR2 at a novel site. The PAR231–72 peptide (10 μm) was incubated with APMA-activated MMP-1 (400 nm; A), MMP-8 (20 nm; B), or MMP-13 (200 nm; C) for 24 h, and reversed-phase HPLC was performed. HPLC chromatograms are representative of at least two independent experiments and are presented as separate graphs for clarity with the same control chromatogram presented in each panel. Peaks identified by HPLC were collected and subjected to further analysis by electrospray MS, which identified MMP-derived cleavage sites at Ser37-Leu38 and Val68-Leu69, to reveal a putative neoepitope-tethered ligand (underlined in D). The colored arrows (A–C) and lines (D) indicate the following: red, parent peptide; green, Ser37-Leu38 cleavage; amber, Val68-Leu69 cleavage; blue, Ser37-Leu38 and Val68-Leu69 cleavage. Observed masses are presented in Table S1. mAU, milli-absorbance units.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Incubation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: The collagenases cleave PAR2 with varying efficiencies. 2-Abz-SKGRSLIG-Y(NO2) peptide (10 μm) was incubated with day 14 conditioned media from IL-1 + OSM-stimulated bovine nasal cartilage explant cultures in the presence or absence of 100 μm GM6001, 10 μm E64, or 2 mm diisopropyl fluorophosphate (DFP). Data (mean ± S.D.) are normalized to the no-inhibitor control sample and are representative of at least two independent experiments with conditioned media from different cartilages (A). 2-Abz-SKGRSLIG-Y(NO2) peptide (50 μm) was incubated with APMA-activated recombinant pro-MMP-1 (50 nm), -8 (10 nm), or -13 (20 nm) in the presence or absence of 50 μm GM6001 or DMSO-only control, and data were normalized to the inhibitor/DMSO negative sample (mean ± S.D.), combining means (each with n = 2 technical replicates) from four independent experiments (B). Michaelis–Menten curves (mean ± S.D.; presented graphs show combined means (each with n = 2 technical replicates) of three independent experiments) were generated using TIMP-1–titrated APMA-activated recombinant pro-MMP-1, -8, and -13 (C). The hydrolysis of substrate was quantified (nm·s−1) using a standard curve determined by total substrate hydrolysis, and nonlinear regression analysis was performed to generate kinetic constants Km and Vmax. kcat was subsequently calculated from Vmax and active enzyme concentration. Tabulated kinetic constants (mean ± S.D.) are from three independent experiments. Matriptase kinetic parameters are included for comparison (D). Selected statistical comparisons were performed using Student's two-tailed unpaired t tests where *** indicates p < 0.001. All error bars represent S.D.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Incubation, Recombinant, Generated, Concentration Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: Activation of PAR2 by the canonical activator peptide SLIGKV-NH2 results in MMP1, MMP13, and ATF3 expression. SW1353-PAR2 cells (blue lines) or empty vector control cells (red lines) loaded with Rhod-4-AM fluorescent calcium probe were subjected to titrations of 0–10 μm SLIGKVD-NH2 (injected at arrow S) followed by 5 μm ionomycin (injected at arrow Io), and calcium mobilization was measured. Data are presented relative to basal fluorescence (at 0 s) and are representative of two independent experiments (each with n = 3 technical replicates) (A). SW1353-PAR2 cells were stimulated with 100 μm SLIGKV-NH2 for the indicated times, and RT-qPCR was performed for ATF3 (B) and MMP1 and MMP13 (C). Data are expressed relative to GAPDH and presented as -fold change compared with basal expression (mean ± S.D., n = 4) and are representative of two independent experiments. SW1353-PAR2 or empty vector control cells were stimulated with 100 μm SLIGKV-NH2 for 48 h, and the conditioned medium was used to perform MMP-1 and MMP-13 ELISAs. Data are presented as mean ± S.D. and are representative of three independent experiments (each with n = 6 technical replicates) (D). Selected statistical comparisons were performed using Student's two-tailed unpaired t tests against basal (unstimulated) where *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05 for MMP1 and ATF3 and ### indicates p < 0.001, ## indicates p < 0.01, and # indicates p < 0.05 for MMP13. All error bars represent S.D. AFU, arbitrary fluorescence units.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Injection, Fluorescence, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: LIGKVD-NH2 is not a canonical PAR2 activator. SW1353-PAR2 cells (blue lines) or empty vector control cells (red lines) loaded with Rhod-4-AM fluorescent calcium probe were subjected to titrations of 0–100 μm LIGKVD-NH2 (left panel) or DVKGIL-NH2 (right panel) and injected at arrow L or D, respectively, followed by 5 μm ionomycin (injected at arrow Io), and calcium mobilization was measured. Data are presented relative to basal fluorescence (at 0 s) and are representative of two independent experiments (each with n = 3 technical replicates) (A). SW1353-PAR2 cells were stimulated with 100 μm SLIGKV-NH2, 100 μm LIGKVD-NH2, 100 μm DVKGIL-NH2, or 50 nm matriptase for 90 min (left panel) or 24 h (right panel), and RT-qPCR was performed for ATF3 or MMP1. Data are expressed relative to GAPDH and presented as -fold change compared with basal expression (mean ± S.D., n = 6) and are representative of three independent experiments (B). SW1353-PAR2 or empty vector control cells were stimulated with 100 μm SLIGKV-NH2 (C), 100 μm LIGKVD-NH2 (D), or 100 μm DVKGIL-NH2 (E) for the indicated times, and then cell lysates were immunoblotted for phospho-ERK1/2 (p-ERK1/2), ERK1/2, phospho-p38 (p-p38), or p38. Combined densitometric scans of three independent experiments (mean ± S.D.) are presented. Statistical comparisons were performed using Student's two-tailed unpaired t tests comparing stimulated cells with basal where *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05. All error bars represent S.D. AFU, arbitrary fluorescence units.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Plasmid Preparation, Injection, Fluorescence, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: MMP-1 is antagonistic to canonical PAR2 activation. SW1353-PAR2 cells were pretreated with either 1000 nm active MMP-1 or serum-free medium for 120 min prior to the addition of 10 nm matriptase or 100 μm SLIGKV-NH2 for an additional 60 min, and RT-qPCR was performed for ATF3 (A). SW1353-PAR2 or empty vector control cells were stimulated with either 1000 nm active MMP-1 or 100 μm SLIGKV-NH2 for 60 min, and RT-qPCR was performed for ATF3 (B). SW1353-PAR2 cells were pretreated with 100 μm LIGKVD-NH2 or serum-free medium for 120 min prior to the addition of 100 μm SLIGKV-NH2 for an additional 60 min, and RT-qPCR was performed for ATF3 (C). SW1353-PAR2 cells were pretreated with 100 μm DVKGIL-NH2 or serum-free medium for 120 min prior to the addition of 100 μm SLIGKV-NH2 for an additional 60 min, and RT-qPCR was performed for ATF3 (D). Data are expressed relative to GAPDH and presented as -fold change compared with basal expression (mean ± S.D., n = 6) and are representative of at least three independent experiments. Selected statistical comparisons were performed using Student's two-tailed unpaired t tests where *** indicates p < 0.001. All error bars represent S.D.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Activation Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

doi: 10.1074/jbc.RA119.006974

Figure Lengend Snippet: Collagenolytic MMPs are induced by PAR2 activation and can antagonize further PAR2 activation. Data presented within this study demonstrate that canonical PAR2 activation is able to induce MMP1 and MMP13 expression and subsequent secretion from chondrocytes, and the addition of MMP-1 to chondrocytes prior to canonical proteolytic stimulation results in an attenuated activation potential of PAR2.

Article Snippet: Lentivirus generation and transduction The lentiviral expression plasmid pSIEW-hPAR2 was constructed using a BamHI-tagged human PAR2 PCR product generated from the human PAR2 VersaClone cDNA (RDC0166, R&D Biosystems) with the primers 5′-AAAAGGATCCGCCACCATGCGGAGCCCCAGC-3′ (forward) and 5′-GCGCGGCCGCGGATCCTCAATAGGAGGTCTTAACAGTGGTTGAAC-3′ (reverse) prior to routine subcloning into BamHI-digested pHR-SINcPPT-SIEW (a generous gift from Prof. Olaf Heidenreich, Newcastle University, UK).

Techniques: Activation Assay, Expressing

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Comprehensive flowchart of adult cardiomyocyte isolation, transfection with siCocktail or siControl, and RNAseq analysis. (B) Bar graph representing the significant knockdown of Rb1 and Meis2 after siRNA transfection. (C) Venn diagram represents the comparative analysis approach to analyze the differentially expressed genes among the groups. (D) Heatmap demonstrates differentially expressed genes after knocking down Rb1 and Meis2 at early and late time points versus control. (E) Sankey and dot plot represents the pathways associated with the up-regulated genes from both early and late-responding groups. (F) Venn diagram showing the 25 genes, commonly up-regulated with ≥2-fold change in siCocktail transfected group (at 48h and D7 after transfection). This analysis further revealed that all 25 genes were downregulated in the control groups at both time points (48h vs D7 post-transfection). (G) Heat map showing the expression of all 25 genes (identified in sub-panel ‘F’) among different study groups. (H) The bar graph shows the expression of MCM2 at early (48h) and late (D7) time points after knocking down Rb1 and Meis2 . (I) The immuno-blot images show reduced expression of MCM2 in adult hearts versus neonatal hearts, and the (J) corresponding bar graph shows the quantification of Western blot. N=6 mice per group. Data represented as mean±SE. * = p-value ≤0.05. Statistical analysis: T -test was used to compare the two groups. P value ≤ 0.05 was considered statistically significant. siCocktail = siRb1+ siMeis2.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Isolation, Transfection, Knockdown, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Comprehensive flowchart representing the hypothesis that inhibition of Rb1 improves cardioprotection through MCM2 . (B) Schematics showing in vivo experimental design, illustrating LAD ligation, intramyocardial injection of MCM2 overexpressing plasmid, or placebo. Animals were observed for 21 days before euthanization and sample collection for molecular and histochemical analysis. Trans- thoracic echocardiography was performed on day 3 and day 21, post-surgery. (C) Dot plot representation of heart-to-body weight ratio among the study groups. (D) Representative echocardiography images showing the cardiac function in different study groups. Dot plots representing (E) ejection fraction, and (F) cardiac output among the study groups. (G) Representative Masson’s trichrome-stained images show cardiac remodeling among the groups. (H) Dot plot representing the quantitative analysis of Masson’s trichrome stained heart sections in all study groups. N=6 mice per group. Data represented as mean±SE. Scale bars represent the magnification of the corresponding image. Statistical analysis: One-way ANOVA was used to compare multiple groups, and post-hoc analysis (Bonferroni test) was performed to correct the p values from multiple comparisons. P value ≤ 0.05 was considered statistically significant. * = p- value ≤0.05. # = statically nonsignificant.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Inhibition, In Vivo, Ligation, Injection, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Representative immunohistology images showing vascular density in different study groups. Vascular density was analyzed through αSMA (green), and vWF (red) staining. (B) The dot plot shows the quantification of measured vascular density per field in different study groups. (C) Schematic representation of hypothesis, showing that the paracrine factors from MCM2 overexpressing adult CMs communicate with endothelial cells and improve angiogenesis. (D) Illustration of tube formation assay, showing ACM transfection and conditioned medium (CM) isolation, which was used to treat HUVECs and the subsequent tube formation assay. (E) Representative bright-field images illustrating endothelial tube formation potential in MCM2-CoMed treated groups versus control. (F-H) Dot plots represent the quantification of various parameters of tube formation. Quantification of endothelial tube formation was performed by Image J. Data represented as mean±SE. N = ≥ 10 images per group. Statistical significance was calculated through a T -test to compare the data between the groups. However, ANOVA was used to compare the data among the groups, and post hoc analysis (Bonferroni test) was performed to correct the p values from multiple comparisons. P value ≤ 0.05 was considered statistically significant. * = p-value ≤0.05. # = statically nonsignificant. Scale bars represent the magnification of the corresponding image. In vivo analysis: N =6 animals per group. In vitro: more than 10 images per group were analyzed for vascular density analysis.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Staining, Tube Formation Assay, Transfection, Isolation, Control, In Vivo, In Vitro

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Schematic illustration of protein-protein interaction analysis using TurboID technology. (B) Silver staining image of a 2D SDS-PAGE showing the enrichment of proteins, interacting with MCM2. (C) Venn diagram revealing 243 proteins, interacting with MCM2. (D) Sankey and dot plot shows the biological pathways associated with the proteins that physically interact with MCM2. The size of the bubble corresponds to the number of genes in that component. Whereas, the color of the bubble corresponds to the significance value. (E) The chord plot demonstrates the MCM2 interacting proteins, which also occur in extracellular space and may contribute to paracrine signaling.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Silver Staining, SDS Page

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: Schematics illustration of MCM2 overexpression in adult CM using AAV9 viral vector, collection of exosomes from MCM2 overexpressing CM, and proteomic analysis. The secretory proteins were characterized through Mass spectrometry. (B) Venn diagram revealed 23 unique proteins, which are secreted from the MCM2 overexpressing CM versus control. (C) The heatmap shows the expression pattern of the MCM2-induced 23 secretory proteins in previous RNAseq data after knocking down Rb1 and Meis2 in adult CM. The expression patterns of all 23 proteins were similar in the RNA seq analysis at early and late time points. (D) In silico analysis shows interaction between the 31 MCM2 interacting proteins with 13 proteins, which are secreted from MCM2 overexpressing CM. (E) In silico analysis using FunRich3.1.3 shows that many of the 23 proteins, which are specifically secreted from MCM2 overexpressing CMs are being regulated by hypoxia and stress-associated transcription factors.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Over Expression, Plasmid Preparation, Mass Spectrometry, Control, Expressing, RNA Sequencing Assay, In Silico

Journal: Molecular cell

Article Title: Native Chromatin Proteomics Reveals Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

doi: 10.1016/j.molcel.2019.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: T4 DNA Polymerase , NEB , Cat# M0203L.

Techniques: Purification, Recombinant, Silver Staining, Protease Inhibitor, Hybridization, Western Blot, Staining, Microscopy, Mass Spectrometry, Software, Real-time Polymerase Chain Reaction