Journal: CNS Neuroscience & Therapeutics

Article Title: Inhibition of ADORA3 promotes microglial phagocytosis and alleviates chronic ischemic white matter injury

doi: 10.1111/cns.14742

Figure Lengend Snippet: Effects of ADORA3 antagonist on WMI and cognitive impairment after BCAS. (A) Grouping of mice and procedure. (B) NOR test ( n = 12). (C) Y maze test ( n = 12). (D) Cue‐related FC test ( n = 12). (E) Context‐related FC test ( n = 12). (F, G) Representative FA images and quantitative analysis ( n = 12). (H–J) Representative western blot images and quantitative analysis of PLP1 and MAG ( n = 5). (K–M) Immunofluorescence staining and quantitative analysis of MBP and LFB staining in the CC, EC, and STR ( n = 5). Scale bar 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following transfer, the membranes were blocked with a 50 g/L skimmed milk solution for 2 h. Primary antibodies were added overnight at 4°C, including ADORA3 (Alomone Labs, AAR‐004, 1:500), PLP1 (Abclonal, A20009, 1:2000), MAG (Abcam, ab89780, 1:2000), Mertk (Abcam, ab95925, 1:1000), Axl (Abcam, ab215205, 1:1000), p‐CREB (Cell Signaling Technology, 9198, 1:1500), CREB (Cell Signaling Technology, 9197, 1:1500), and GAPDH (Proteintech, 60004‐1‐ig, 1:1000).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Inhibition of ADORA3 promotes microglial phagocytosis and alleviates chronic ischemic white matter injury

doi: 10.1111/cns.14742

Figure Lengend Snippet: H‐89 reverses the protective effect of MRS1523 against WMI and cognitive impairment after BCAS. (A) Grouping of mice and procedure. (B) NOR test ( n = 12). (C) Y maze test ( n = 12). ( D ) Cue‐related FC test ( n = 12). (E) Context‐related FC test ( n = 12). (F, G) Representative FA images and quantitative analysis ( n = 12). (H–J) Representative western blot images and quantitative analysis of PLP1 and MAG ( n = 5). (K–M) Immunofluorescence staining and quantitative analysis of MBP and LFB staining in the CC, EC, and STR ( n = 5). Scale bar 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following transfer, the membranes were blocked with a 50 g/L skimmed milk solution for 2 h. Primary antibodies were added overnight at 4°C, including ADORA3 (Alomone Labs, AAR‐004, 1:500), PLP1 (Abclonal, A20009, 1:2000), MAG (Abcam, ab89780, 1:2000), Mertk (Abcam, ab95925, 1:1000), Axl (Abcam, ab215205, 1:1000), p‐CREB (Cell Signaling Technology, 9198, 1:1500), CREB (Cell Signaling Technology, 9197, 1:1500), and GAPDH (Proteintech, 60004‐1‐ig, 1:1000).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Conformational Antibodies to Proteolipid Protein-1 and Its Peripheral Isoform DM20 in Patients With CNS Autoimmune Demyelinating Disorders

doi: 10.1212/NXI.0000000000200359

Figure Lengend Snippet: (A) Monoclonal antibodies against different epitopes of PLP1 and serum from a patient with ADD label the surface of live HEK293T cells transfected with PLP1-EGFP-tag (green). Merged images show colocalization of the staining (orange). No binding is observed with the MOG 8-18C5 monoclonal antibody or with serum from a healthy control. (B) Structure of PLP1. Colored aminoacidic sequences identify the different epitopes recognized by the monoclonal antibodies (yellow = PLP1 50-69 ; green = PLP1 178-191 , and blue = PLP1 200-219 ) or their intracellular portion, which is absent in the DM20 protein isoform (red). (C) Serum of a PLP1-IgG positive patient (green) binds to the myelinated areas (cerebellum, striatum and hippocampus) of lightly fixed rat brain sagittal slices (indirect immunofluorescence). PLP1 monoclonal antibody (red) binds to the same regions, colocalizing with patients' serum (yellow). DAPI stains cell nuclei. Scale bar: 50 µm. (D) Serum from the same patient stains both PLP1-CBA and TBA on lightly fixed rat brain (left column); unmodified staining after preadsorption on MOG-transfected cells (central column); abolition of the binding after preadsorption on PLP1-transfected cells on both CBA and TBA (right column). Created in BioRender. Gastaldi, M. (2024) BioRender.com/v24o621 . ADD = autoimmune demyelinating disorder; CBA = cell-based assay; MOG = myelin oligodendrocyte glycoprotein; PLP1 = proteolipid protein-1; TBA = tissue-based assay.

Article Snippet: Live cell-based assays were implemented using the following plasmids: EGFP-tagged full-length human MOG and untagged rat full-length MOG (kind gift of Prof M. Reindl); GFP-tagged human PLP1 (code: RG218616, Origene); untagged human PLP1 (code: SC119823, Origene); untagged human DM20 (a splicing variant of PLP1, code: SC121107, Origene).

Techniques: Bioprocessing, Transfection, Staining, Binding Assay, Control, Immunofluorescence, Cell Based Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Conformational Antibodies to Proteolipid Protein-1 and Its Peripheral Isoform DM20 in Patients With CNS Autoimmune Demyelinating Disorders

doi: 10.1212/NXI.0000000000200359

Figure Lengend Snippet: (A) Serum of a patient with PLP1-IgG binds to DM20-transfected CBA (green) and colocalizes with a PLP1 mAb (red) directed against an extracellular loop of the protein (top row). No binding is observed with serum from a HC (bottom row). (B) Binding to both PLP1- and DM20-transfected cells (green) is observed with both PLP1mAb (first row) and serum from a patient with CNS+PNS ADD (pt#1; second row). Conversely, serum from a patient with CNS ADD binds exclusively to PLP1-transfected cells and not to DM20-transfected cells (third row). No binding is observed with serum from a HC (bottom row). DAPI stains the nuclei. (C) Heatmap representing fluorescence intensity of the available samples (n = 36) in either PLP1 or DM20 CBA measured with a semiquantitative score. All PLP1-IgG–positive patients with CNS + PNS ADD, unlike patients with other disease groups, were positive on both PLP1 and DM20 CBA. Scale bars: 10 µm. ADD = autoimmune demyelinating disorder; CBA = cell-based assay; Ig = immunoglobulin; HC = healthy control; mAbs = monoclonal antibodies; PNS = peripheral nervous system; PLP1 = proteolipid protein-1; Pt = patient.

Article Snippet: Live cell-based assays were implemented using the following plasmids: EGFP-tagged full-length human MOG and untagged rat full-length MOG (kind gift of Prof M. Reindl); GFP-tagged human PLP1 (code: RG218616, Origene); untagged human PLP1 (code: SC119823, Origene); untagged human DM20 (a splicing variant of PLP1, code: SC121107, Origene).

Techniques: Transfection, Binding Assay, Fluorescence, Cell Based Assay, Control, Bioprocessing

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A) Translocation with der(4) and der(12). The hand roentgenogram, especially the shortened metacarpals of digits 4 and 5, are diagnostic of BDE. (B) 6C. Formaldehyde-crosslinked chromatin of C28/I2 chondrocytes was sonicated. After ChIP, the chromatin was blunt-end repaired and ligated at ultra-high dilution to favor intramolecular ligation events. Chromatin loops were PCR amplified with inverse oligos, and the amplicons were subcloned and sequenced. PTHLH and SOX9 promoters were the bait sequences. (C) Example of PTHLH 6C. Several bp were inserted due to the end repair of chromatin fragments. The interacting sequence was located at 52,431,496 bp on chromosome 12 (positions in bp; UCSC assembly hg18). In trans event in SOX9 6C. The interacting element on chromosome 12 was identified next to the bait sequence of the SOX9 promoter. (D) Restrictive classification of the 6C sequence reads. Either the promoter (bait) or the promoter interaction with any sequence was counted. (E) PTHLH and SOX9 mRNA expression in human C28/I2 and LCL.

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: Translocation Assay, Diagnostic Assay, Sonication, Ligation, Amplification, Sequencing, Expressing

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A and B) Interaction frequencies of SOX9 (A) and PTHLH (B) 6C data. In SOX9 6C, 4 CREs were detected; 9 were found in PTHLH 6C (black symbols). re70373, re18527, and re52431 were the most abundant, and further characterized by high mammalian conservation, H3K4me1 enrichment (ENCODE), and ESPERR regulatory potential (denoted by “+”) (positions in bp; UCSC assembly hg18). re52431 interacted with SOX9 in trans. (C) New 6C libraries validated prior 6C results; control was a LCL. A direct PCR approach amplified re70373, re18527, and re52431. In the LCL, the C28/I2 CRE interactions were significantly reduced; only for re70373 were interactions in C28/I2 and LCL detected equally. (D) Distances of cis- and trans-regulatory elements identified in PTHLH 6C and SOX9 6C. re18527 (18,527,200 bp) and re52431 (52,431,500 bp) interacted with PTHLH, re70373 (70,373,400 bp) interacted in cis and re52431 in trans with SOX9. re52431 was further named CISTR-ACT, due to its cis and trans interactions. (E) Luciferase reporter assays of re70373, CISTR-ACTF (full-length regulatory sequence), and CISTR-ACTS (containing the most conserved part) elements were placed in front of the SOX9 promoter; all 3 enhanced transcription. (F) re18527, CISTR-ACTF, and CISTR-ACTS, either alone or in combination, controlled the PTHLH promoter and enhanced luciferase transcription (n = 6). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: Control, Amplification, Luciferase, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A) Examples of colocalized vs. separate signal pairs in C28/I2 nuclei (distances in μm; scale bars: 2 μm). (B) Accumulated colocalized signals were found in all experiments. Shown are genomic distances between probes (in Mb); Mann-Whitney test was used to determine significance. PTHLH was in physical proximity to re18527 compared with the re18527 negative control, with a 9.5-Mb equidistant upstream probe 12p(1). (C) CISTR-ACT significantly showed interaction with PTHLH compared with the 3 controls. PTPLAD1 was not expressed in C28/I2. (D) re70373 was marginally in spatial proximity to SOX9. (E) Significant in trans actions of CISTR-ACT with SOX9. In the control of SOX9 with a chromosome 15q(1) probe, only 2 signal pairs had distances less than 1 μm.

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: MANN-WHITNEY, Negative Control, Control

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A) CISTR-ACTS and CISTR-ACTF, with or without re18527, were cloned in a promoter and regulator lacking plasmid (pGl2-luci). In CISTR-ACTF–transfected C28/I2 cells, DA125942 was highly expressed. re18527 blocked the transcription; CISTR-ACTS produced no transcription. (B) SOX9 expression was knocked down relative to the scrambled siRNA control. Depleted PTHLH or DA125942 downregulated SOX9. siRNA-mediated depletion of PTHLH and SOX9 upregulated DA125942, and PTHLH expression was significantly reduced by PTHLH, DA125942, and SOX9 siRNA (n = 6). (C) Overexpression of PTHLH, SOX9, and DA125942 confirmed the expression-dependent network (n = 4). (D) STRING network. Expression array analysis revealed 22 organ morphogenesis genes (GO:0009887) showing interactions. Line thickness is indicative of physical or functional interaction confidence. Mesenchymal and prechondrogenic genes were differentially regulated. (E and F) Fibroblasts of affected BDE patients (AFF) and 3 nonaffected subjects (NON) were chondrogenically induced. DA125942 was upregulated in patients of both BDE families (n = 3). (G) RNA-ChIRP on C28/I2, nonaffected subject, and BDE patient chromatin. DA125942 was retrieved in contrast to GAPDH. (H and I) ChIRP on eluted C28/I2 DNA detected DA125942 binding at the PTHLH and SOX9 loci. Each amplicon was mapped by the UCSC custom tracks (numbered black boxes). (J and K) In t(4;12) and t(8;12), reduced lncRNA occupancy at the PTHLH locus was observed; binding at SOX9 was not affected (n = 2). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Produced, Expressing, Control, Over Expression, Functional Assay, Binding Assay, Amplification

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A and B) Phylogenetic analysis of the CISTR-ACT DNA and lncRNA sequences. 121 conserved TFBSs were bioinformatically predicted. The differences of the scales indicate that the lncRNAs are evolutionarily more divergent than the conserved DNA sequences. (C) Genomic arrangement of murine and rat Pthlh, Sox9, and CISTR-ACT–encoded lncRNAs AW491522 and BM385392, respectively, suggested an in trans network. (D) siRNA transfections of mouse ATDC5 (10 days differentiated) chondrocytes. Depletion of the mouse lncRNA AW491522 caused Pthlh or Sox9 downregulation. Knockdown of either Pthlh or Sox9 depleted the lncRNA (n = 4). (E) siRNA assays in RCSs. An analogous expression-dependent network was observed compared with the ATDC5 results (n = 5).**P ≤ 0.01.

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: Transfection, Knockdown, Expressing

Journal: The Journal of Clinical Investigation

Article Title: A misplaced lncRNA causes brachydactyly in humans

doi: 10.1172/JCI65508

Figure Lengend Snippet: (A) AW491522 and BM385392 retrieval. Mouse or rat Gapdh was not precipitated. (B and C) ChIRP on eluted DNA detected AW491522 binding at the murine Pthlh (B) and Sox9 (C) loci. Each amplicon was mapped by the UCSC custom tracks (numbered black boxes). (D and E) BM385392 binding at the rat Pthlh (D) and Sox9 (E) loci. The lncRNA’s occupancy was observed at homologous regions in mouse and rat (red shading). Annotated mammalian conservation is shown; genomic positions correspond to mouse assembly mm9 and rat assembly rn4 (n = 2).

Article Snippet: PTHLH (SC307833) and SOX9 (SC321884) expression plasmids were obtained from OriGene.

Techniques: Binding Assay, Amplification