Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A, B) Force-directed graph of VSMCs isolated 5 days after injury showing Runx1 expression (A) or the result of RUNX1 overexpression simulation (B) . (C) Direct and indirect targets of Runx1 in the PrP GRN showing differential expression between PrP and Non-RSP cells on a blue (higher in Non-RSP) to red (higher in PrP) colour scale. Black borders indicate significantly differential expression (p adj <0.05). Edge widths reflect edge connectivity and edge colour show positive (red) and negative (blue) interactions. (D) Mmp14 transcript levels detected by quantitative RT-PCR in mVSMCs transfected with non-targeting (NTC) or Runx1 -targeting siRNA (Runx1-siRNA), or transduced with an empty vector (CTRL-EV) or RUNX1-overexpressing (OE) lentivirus. Dots represent cells from independent animals (N=5) and lines indicate mean expression. (E) Schematic of clonal proliferation assay where a red fluorescent protein (RFP)-expressing lentivirus is used to test the effect of RUNX1 cDNA relative to an empty control vector (EV) on the ability of lineage-labelled VSMCs from Myh11-EYFP animals to form colonies (left). Right panel shows the percentage of cells forming a clonal VSMC patch in RUNX1-OE and empty vector control cells (CTRL-EV). (F) Fold change in %EdU+ cells after siRNA-mediated RUNX1 depletion (RUNX1-siRNA, relative to non-targeting siRNA-treated cells) and lentivirus-mediated RUNX1-overexpression of hVSMCs (RUNX1-OE, relative to cells transduced with empty vector virus). (G-I) Representative immunostaining of non-plaque aorta (G and H, N=10) or carotid endarterectomy samples (I, N=6) for RUNX1, αSMA and KI67 as indicated. Scale bars: G: 20 µm, H: 100 µm (left, middle) and 20 µm (right), I: 50 µm. FC: fibrous cap, L: Lumen, I: Intima, M: Media. Examples of RUNX1+ (closed arrowheads) and RUNX1-cells (open arrowheads) that are αSMA+ are indicated. Asterisk indicates p<0.05.

Article Snippet: Sequential sections were either H&E stained, or co-stained for αSMA (DAKO, M0851), detected with biotin-coupled anti-Mouse (DAKO, E0433) and either RUNX1 (clone A-2, Santa Cruz, sc-365644), KI67 (Cell Signaling Technology, 8114), TIMP1 (clone F31 P2 A5, ThermoFisher, MA1-773), or CD74 (clone LN-2, Santa Cruz, sc-6262) that were detected with HRP-conjugated anti-Rabbit (Cell Signaling Technology, 8114, using DAB peroxidase substrate (SignalStain)).

Techniques: Isolation, Expressing, Over Expression, Quantitative Proteomics, Quantitative RT-PCR, Transfection, Transduction, Plasmid Preparation, Proliferation Assay, Control, Virus, Immunostaining

Journal: bioRxiv

Article Title: AP-4 mediated ATG9A sorting underlies axonal and autophagosome biogenesis defects in a mouse model of AP-4 deficiency syndrome

doi: 10.1101/235101

Figure Lengend Snippet: A. Schematic of the KO tm1b allele, showing removal of critical exon 3. B. Representative genotyping PCR of litter used for E16 hippocampal neuronal culture showing AP-4ε WT (+/+) , KO (-/-) and Het (+/-) embryos. Bottom panel, western blot showing loss of ε protein in KO embryos. C. ε protein level in brain regions of adult mice (n = 3 repeats). D. E. Sections prepared from animals at P30 stained against NeuN and GFAP showing lateral ventricular enlargement in KO. Scale bar = 200 μm. (E) Quantification of relative area of lateral ventricle (n = 5 animals WT/KO). F - H. Commissural crossing axons, stained against Neurofilament-200 (NF200). Scale bar = 100 um. (G) Quantification of thickness of CC and (H) DF (n = 5 animals WT/KO). Quantified data is expressed as mean ± SEM. (E) presented relative to control value (n = 5 animals per genotype). Statistical analysis: Two-tailed unpaired Student’s t-test, p<0.05, **P< 0.01 and ***P<0.001. CC - corpus callosum, DF - dorsal fornix.

Article Snippet: For immunocytochemistry (ICC), Immunohistochemistry (IHC) and western blotting (WB) antibodies were used with the following dilutions; Actin (Sigma A2066; WB: 1/1,000), AP-4ε (BD Biosciences 612018; WB: 1/300, ICC: 1/250), ATG9A (Rabbit STO-219 WB: 1/2000, IF: 1/2000 ( )), ATG9A (Hamster 14F2 8B1; IF: 1/500 ( )), GFP (Nescalai Tesque 04404-84; ICC: 1/1000), GFAP (Dako Z0334; ICC: 1/300), GM130 (BD Biosciences 610822; ICC: 1/1000), Golgin-97 (CST 13193; ICC: 1/250), MAP2 (Synaptic Systems 188-004, ICC: 1/500), NeuN (Chemicon MAB377; IHC: 1/300), NF200 (Abcam ab4680; IF: 1/500 IHC: 1/500) and MYC (NeuroMab 9E10; WB: 1/100, ICC: 1/100).

Techniques: Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: RNA-programmable cell type monitoring and manipulation in the human cortex with CellREADR

doi: 10.1101/2024.12.03.626590

Figure Lengend Snippet: A) Human organotypic slice model. Human neocortical tissue specimens (here, middle and inferior temporal gyri; left) were sectioned into 350µm slices and cultured on semi-permeable membranes for up to 16 days in vitro (DIV; right). CellREADR AAV vectors were directly applied to slices on DIV 0. B) Schematic of CellREADR design. In the single virus design (left), binding of the sensor RNA sequence to the target leads to ADAR-mediated editing of a stop codon positioned between the sensor and the effector, leading to expression of the downstream effector (here, mNeon). The binary system (right) uses a CellREADR virus (top right) to drive expression of tTA2 (Tet-Advanced transactivator) in the target cell. A reporter virus, designed to conditionally express a selected effector molecule (e.g. mNeon, ChIEF, GCamp7f, depending on experiment) under control of the Tet-Responsive Element (TRE), is also applied. C) CellREADR targeting of diverse neuronal populations. Binary CellREADRs (mNeonGFP reporter) designed against Somatostatin ( SST ), Unc-5 netrin receptor B ( UNC5B), Parvalbumin ( PVALB ), Forkhead box protein P2 ( FOXP2) , and Calretinin ( CALB2 ) were applied to slices of human neocortex. Slices were immunostained for mNeon 7 days after virus application. The CellREADR binary system led to robust expression of mNeon in differing laminar distributions and in cells of various morphologies. Dashed red boxes highlight the area depicted in the accompanying magnified image; pia and white matter (WM) are illustrated with dotted lines. D) Histological analysis of FOXP2 and CALB2 CellREADR targeting. CellREADR mNeon distribution (green, left) and protein expression of target (purple, middle) are depicted at DIV 7. The fluorescence intensity profile to the left of each image set reveals the distribution of cells targeted by the CellREADR. Dashed red boxes indicate areas depicted in 1F; pia and white matter (WM) are illustrated with dotted lines. E) Localization of CellREADR-targeted cells. CellREADR mNeon fluorescence intensity profiles measured across the depth of the cortex (0% depth = pial surface, 100% depth = white matter boundary) in 6 slices from 3 subjects. FOXP2 -mNeon was relatively uniform throughout cortical layers, while CALB2 -mNeon was more restricted to outer cortical layers. F) Immunohistochemical characterization of CellREADR specificity. Representative images showing colocalization of CellREADR-mNeon with the corresponding target. G) Quantification of CellREADR specificity, as measured by immunostaining. Each circular point denotes the specificity of labeling measured from an individual subject’s tissue. Horizontal bars indicate mean specificity values for the CellREADRs. Throughout figures, data are presented as mean ± SEM. H) Benchmarking of CellREADR efficiency with a human interneuron enhancer virus, DLX2.0-YFP. CALB2 CellREADR and DLX2.0-YFP viruses (rAAV2-retro) were applied to slices cut sequentially from the same neocortical tissue specimen. Tissue was fixed 7 days after virus application, and immunostained against mNeon and YFP. Slice boundaries are indicated by the solid white line, and the white matter is demarcated by a dashed white line; colored boxes mark the inset images below. I) Localization of DLX2.0-YFP expression. Representative image illustrating the efficiency and distribution of cellular labeling achieved with a DLX2.0-YFP virus (left), with fluorescence intensity profile (right) illustrating distribution of cells labeled by the virus. As compared to CALB2 CellREADR labeling ( and ), DLX2.0-YFP+ cells were less concentrated in the outer cortex, as would be expected for pan-interneuron targeting by DLX2.0. J) Ability of DLX2.0-YFP to label the CALB2+ population. Quantification of colocalization of DLX2.0-YFP expression and CALB2, as measured by immunostaining (images not shown); note inter-subject variability in targeting of CALB2 cells. K) Efficiency of CALB2 CellREADR and DLX2.0-YFP viruses. Quantification of cells labeled per unit area. Note, although the CALB2 CellREADR is designed to target a subset of interneurons, rather than multiple classes of interneurons (as expected for DLX2.0), it actually labeled more cells, suggesting that it was more efficient.

Article Snippet: The following antibodies were used for immunohistochemical labeling of mNeon, mCherry, FoxP2, Calretinin and NeuN: mouse anti-mNeon Ab 1:500, Proteintech 32f6; chicken anti-mCherry Ab 1:500, Origene TA150127; rabbit anti-FoxP2 Ab 1:500, Abcam ab16046; rabbit anti-Calretinin Ab 1:500, Swant CR7697; mouse anti-NeuN Ab 1:500, Cell Signaling #94403S.

Techniques: Cell Culture, In Vitro, Virus, Binding Assay, Sequencing, Expressing, Control, Fluorescence, Immunohistochemical staining, Immunostaining, Labeling

Journal: Frontiers in Oncology

Article Title: HER2 expression in urothelial carcinoma, a systematic literature review

doi: 10.3389/fonc.2022.1011885

Figure Lengend Snippet: Overview of HER2+ across all studies.

Article Snippet: Rink 2012 , Observational , , 22 , 18.2% , IHC 3+; CTC Veridex CellSearch tumor phenotyping reagent HER2/neu (IHC) , .

Techniques: Staining, Amplification, Clinical Proteomics